I know PCR can be used to amplify a tiny sample of DNA in order to perform experiments. Is there a similar technique to use on a protein sample? More specifically, I'm not interested in "cutting" up the protein but simply making more of the original sample.
There isn't any technique with one exception. PCR generates DNA from DNA so you can establish cycles. For protein synthesis, an mRNA is a template to produce proteins. Most likely proteins you are interested in would not synthesize mRNAs, so that you can not establish cycles.
The exception I said earlier is Protein Misfolding Cyclic Amplification (PMCA). Some disease associated misfold proteins can induce misfolding of the same polypeptide which is not misfolded. So you can establish cycles of misfolding reaction.
There are technique to synthesize proteins from DNAs or RNAs, but they are not chain reactions. https://www.promega.com/products/protein-expression/eukaryotic-cell-free-protein-expression/ https://www.neb.com/products/e6800-purexpress-invitro-protein-synthesis-kit
It is impossible to amplify protein like PCR. DNA and RNA can undergo PCR because basepairs are complementary: A-T C-G A-U. But amino acids are not complementary; can you find an amino acid that matches glycine? Besides, proteins are not only a single polypeptide chain like DNA, proteins need to be folded after polypeptide chain are synthesized. Usually a protein molecule need more than one polypeptide chain to combine together to form a protein molecule. It can not happen that it works as PCR.
I would say impossible is a stretch. An inventive biochemist could create molecules that are "complimentary" to each amino acid similar to a Dna polymerase. Then a protein would be needed to both detect and translate this similarity into protein elongation. Unlikely, hell yes, impossible, no.
Distinguish among the basic techniques used to manipulate DNA and RNA
- The first step to study or work with nucleic acids includes the isolation or extraction of DNA or RNA from cells.
- Gel electrophoresis depends on the negatively-charged ions present on nucleic acids at neutral or basic pH to separate molecules on the basis of size.
- Specific regions of DNA can be amplified through the use of polymerase chain reaction for further analysis.
- Southern blotting involves the transfer of DNA to a nylon membrane, while northern blotting is the transfer of RNA to a nylon membrane these techniques allow samples to be probed for the presence of certain sequences.
Molecular Analysis of DNA
In this subsection, we will outline some of the basic methods used for separating and visualizing specific fragments of DNA that are of interest to a scientist. Some of these methods do not require knowledge of the complete sequence of the DNA molecule. Before the advent of rapid DNA sequencing, these methods were the only ones available to work with DNA, but they still form the basic arsenal of tools used by molecular geneticists to study the body’s responses to microbial and other diseases.
Nucleic Acid Probing
DNA molecules are small, and the information contained in their sequence is invisible. How does a researcher isolate a particular stretch of DNA, or having isolated it, determine what organism it is from, what its sequence is, or what its function is? One method to identify the presence of a certain DNA sequence uses artificially constructed pieces of DNA called probes. Probes can be used to identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically. For example, DNA probes are used to detect the vaginal pathogens Candida albicans, Gardnerella vaginalis, and Trichomonas vaginalis.
To screen a genomic library for a particular gene or sequence of interest, researchers must know something about that gene. If researchers have a portion of the sequence of DNA for the gene of interest, they can design a DNA probe, a single-stranded DNA fragment that is complementary to part of the gene of interest and different from other DNA sequences in the sample. The DNA probe may be synthesized chemically by commercial laboratories, or it may be created by cloning, isolating, and denaturing a DNA fragment from a living organism. In either case, the DNA probe must be labeled with a molecular tag or beacon, such as a radioactive phosphorus atom (as is used for autoradiography) or a fluorescent dye (as is used in fluorescent in situ hybridization, or FISH), so that the probe and the DNA it binds to can be seen (Figure 1). The DNA sample being probed must also be denatured to make it single-stranded so that the single-stranded DNA probe can anneal to the single-stranded DNA sample at locations where their sequences are complementary. While these techniques are valuable for diagnosis, their direct use on sputum and other bodily samples may be problematic due to the complex nature of these samples. DNA often must first be isolated from bodily samples through chemical extraction methods before a DNA probe can be used to identify pathogens.
Figure 1. DNA probes can be used to confirm the presence of a suspected pathogen in patient samples. This diagram illustrates how a DNA probe can be used to search for a gene of interest associated with the suspected pathogen.
Clinical Focus: Karni, Part 2
This example continues Karni’s story that started in Microbes and the Tools of Genetic Engineering.
The mild, flu-like symptoms that Karni is experiencing could be caused by any number of infectious agents. In addition, several non-infectious autoimmune conditions, such as multiple sclerosis, systemic lupus erythematosus (SLE), and amyotrophic lateral sclerosis (ALS), also have symptoms that are consistent with Karni’s early symptoms. However, over the course of several weeks, Karni’s symptoms worsened. She began to experience joint pain in her knees, heart palpitations, and a strange limpness in her facial muscles. In addition, she suffered from a stiff neck and painful headaches. Reluctantly, she decided it was time to seek medical attention.
- Do Karni’s new symptoms provide any clues as to what type of infection or other medical condition she may have?
- What tests or tools might a health-care provider use to pinpoint the pathogen causing Karni’s symptoms?
We’ll return to Karni’s example later on this page.
Agarose Gel Electrophoresis
There are a number of situations in which a researcher might want to physically separate a collection of DNA fragments of different sizes. A researcher may also digest a DNA sample with a restriction enzyme to form fragments. The resulting size and fragment distribution pattern can often yield useful information about the sequence of DNA bases that can be used, much like a bar-code scan, to identify the individual or species to which the DNA belongs.
Gel electrophoresis is a technique commonly used to separate biological molecules based on size and biochemical characteristics, such as charge and polarity. Agarose gel electrophoresis is widely used to separate DNA (or RNA) of varying sizes that may be generated by restriction enzyme digestion or by other means, such as the PCR (Figure 2).
Due to its negatively charged backbone, DNA is strongly attracted to a positive electrode. In agarose gel electrophoresis, the gel is oriented horizontally in a buffer solution. Samples are loaded into sample wells on the side of the gel closest to the negative electrode, then drawn through the molecular sieve of the agarose matrix toward the positive electrode. The agarose matrix impedes the movement of larger molecules through the gel, whereas smaller molecules pass through more readily. Thus, the distance of migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling a longer distance through the gel. Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run on the same gel. To separate very large DNA fragments, such as chromosomes or viral genomes, agarose gel electrophoresis can be modified by periodically alternating the orientation of the electric field during pulsed-field gel electrophoresis (PFGE). In PFGE, smaller fragments can reorient themselves and migrate slightly faster than larger fragments and this technique can thus serve to separate very large fragments that would otherwise travel together during standard agarose gel electrophoresis. In any of these electrophoresis techniques, the locations of the DNA or RNA fragments in the gel can be detected by various methods. One common method is adding ethidium bromide, a stain that inserts into the nucleic acids at non-specific locations and can be visualized when exposed to ultraviolet light. Other stains that are safer than ethidium bromide, a potential carcinogen, are now available.
Figure 2. Click for a larger image. (a) The process of agarose gel electrophoresis. (b) A researcher loading samples into a gel. (c) This photograph shows a completed electrophoresis run on an agarose gel. The DNA ladder is located in lanes 1 and 9. Seven samples are located in lanes 2 through 8. The gel was stained with ethidium bromide and photographed under ultraviolet light. (credit a: modification of work by Magnus Manske credit b: modification of work by U.S. Department of Agriculture credit c: modification of work by James Jacob)
Restriction Fragment Length Polymorphism (RFLP) Analysis
Restriction enzyme recognition sites are short (only a few nucleotides long), sequence-specific palindromes, and may be found throughout the genome. Thus, differences in DNA sequences in the genomes of individuals will lead to differences in distribution of restriction-enzyme recognition sites that can be visualized as distinct banding patterns on a gel after agarose gel electrophoresis. Restriction fragment length polymorphism (RFLP) analysis compares DNA banding patterns of different DNA samples after restriction digestion (Figure 3).
Figure 3. RFLP analysis can be used to differentiate DNA sequences. In this example, a normal chromosome is digested into two fragments, whereas digestion of a mutated chromosome produces only one fragment. The small red arrows pointing to the two different chromosome segments show the locations of the restriction enzyme recognition sites. After digestion and agarose gel electrophoresis, the banding patterns reflect the change by showing the loss of two shorter bands and the gain of a longer band. (credit: modification of work by National Center for Biotechnology Information)
RFLP analysis has many practical applications in both medicine and forensic science. For example, epidemiologists use RFLP analysis to track and identify the source of specific microorganisms implicated in outbreaks of food poisoning or certain infectious diseases. RFLP analysis can also be used on human DNA to determine inheritance patterns of chromosomes with variant genes, including those associated with heritable diseases or to establish paternity.
Forensic scientists use RFLP analysis as a form of DNA fingerprinting, which is useful for analyzing DNA obtained from crime scenes, suspects, and victims. DNA samples are collected, the numbers of copies of the sample DNA molecules are increased using PCR, and then subjected to restriction enzyme digestion and agarose gel electrophoresis to generate specific banding patterns. By comparing the banding patterns of samples collected from the crime scene against those collected from suspects or victims, investigators can definitively determine whether DNA evidence collected at the scene was left behind by suspects or victims.
Southern Blots and Modifications
Several molecular techniques capitalize on sequence complementarity and hybridization between nucleic acids of a sample and DNA probes. Typically, probing nucleic-acid samples within a gel is unsuccessful because as the DNA probe soaks into a gel, the sample nucleic acids within the gel diffuse out. Thus, blotting techniques are commonly used to transfer nucleic acids to a thin, positively charged membrane made of nitrocellulose or nylon. In the Southern blot technique, developed by Sir Edwin Southern in 1975, DNA fragments within a sample are first separated by agarose gel electrophoresis and then transferred to a membrane through capillary action (Figure 4). The DNA fragments that bind to the surface of the membrane are then exposed to a specific single-stranded DNA probe labeled with a radioactive or fluorescent molecular beacon to aid in detection. Southern blots may be used to detect the presence of certain DNA sequences in a given DNA sample. Once the target DNA within the membrane is visualized, researchers can cut out the portion of the membrane containing the fragment to recover the DNA fragment of interest.
Figure 4. In the Southern blot technique, DNA fragments are first separated by agarose gel electrophoresis, then transferred by capillary action to a nylon membrane, which is then soaked with a DNA probe tagged with a molecular beacon for easy visualization.
Variations of the Southern blot—the dot blot, slot blot, and the spot blot—do not involve electrophoresis, but instead concentrate DNA from a sample into a small location on a membrane. After hybridization with a DNA probe, the signal intensity detected is measured, allowing the researcher to estimate the amount of target DNA present within the sample.
A colony blot is another variation of the Southern blot in which colonies representing different clones in a genomic library are transferred to a membrane by pressing the membrane onto the culture plate. The cells on the membrane are lysed and the membrane can then be probed to determine which colonies within a genomic library harbor the target gene. Because the colonies on the plate are still growing, the cells of interest can be isolated from the plate.
In the northern blot, another variation of the Southern blot, RNA (not DNA) is immobilized on the membrane and probed. Northern blots are typically used to detect the amount of mRNA made through gene expression within a tissue or organism sample.
Another technique that capitalizes on the hybridization between complementary nucleic acid sequences is called microarray analysis. Microarray analysis is useful for the comparison of gene-expression patterns between different cell types—for example, cells infected with a virus versus uninfected cells, or cancerous cells versus healthy cells (Figure 5).
Typically, DNA or cDNA from an experimental sample is deposited on a glass slide alongside known DNA sequences. Each slide can hold more than 30,000 different DNA fragment types. Distinct DNA fragments (encompassing an organism’s entire genomic library) or cDNA fragments (corresponding to an organism’s full complement of expressed genes) can be individually spotted on a glass slide.
Once deposited on the slide, genomic DNA or mRNA can be isolated from the two samples for comparison. If mRNA is isolated, it is reverse-transcribed to cDNA using reverse transcriptase. Then the two samples of genomic DNA or cDNA are labeled with different fluorescent dyes (typically red and green). The labeled genomic DNA samples are then combined in equal amounts, added to the microarray chip, and allowed to hybridize to complementary spots on the microarray.
Hybridization of sample genomic DNA molecules can be monitored by measuring the intensity of fluorescence at particular spots on the microarray. Differences in the amount of hybridization between the samples can be readily observed. If only one sample’s nucleic acids hybridize to a particular spot on the microarray, then that spot will appear either green or red. However, if both samples’ nucleic acids hybridize, then the spot will appear yellow due to the combination of the red and green dyes.
Although microarray technology allows for a holistic comparison between two samples in a short time, it requires sophisticated (and expensive) detection equipment and analysis software. Because of the expense, this technology is typically limited to research settings. Researchers have used microarray analysis to study how gene expression is affected in organisms that are infected by bacteria or viruses or subjected to certain chemical treatments.
Figure 5. (a) The steps in microarray analysis are illustrated. Here, gene expression patterns are compared between cancerous cells and healthy cells. (b) Microarray information can be expressed as a heat map. Genes are shown on the left side different samples are shown across the bottom. Genes expressed only in cancer cells are shown in varying shades of red genes expressed only in normal cells are shown in varying shades of green. Genes that are expressed in both cancerous and normal cells are shown in yellow.
Think about It
- What does a DNA probe consist of?
- Why is a Southern blot used after gel electrophoresis of a DNA digest?
We applied the RPA process to a wide variety of targets in complex DNA templates. The versatility and specificity of the technology are exemplified by the amplification of three genetic markers, apolipoprotein B (apoB), sex-determining region Y (Sry), and porphobilinogen deaminase (PBDG), from complex human genomic DNA (Figure 2A). While the negative controls did not produce a signal, clean amplification products of the correct identity ( Figure S4) were generated in each template-containing sample.
(A) Acrylamide gel electrophoresis of RPA products using primers for three human markers (apoB, Sry, PBDG). Water (−) or 1,000 copies of genomic DNA (+) served as template. Ten percent of each reaction is loaded on the gel. The expected product sizes are 305, 371, and 353 base-pairs for apoB, Sry, and PBDG, respectively.
(B) Real-time RPA using primers for the B. subtilis SpoB locus. Fluorescence upon intercalation of SybrGreenI into nascent product is detected. B. subtilis DNA served as template in triplicate reactions at 10 5 (black), 10 4 (red), 10 3 (yellow), or 100 copies (green) or water (blue). The onset of amplification depends linearly on the logarithm of the starting template copy number [see inset time in minutes (midpoint of growth curve) versus log
The progression of RPA reactions can be monitored in real-time by the inclusion of a sensitive nucleic acid dye (Figure 2B) . Here, primers for a locus in the B. subtilis genome have been used. The amplification of DNA proved to be exponential over a wide range of template concentrations and results were obtained in less than 30 min. The onset of amplification depends linearly on the logarithm of the starting number of template copies. Reactions carried out in the absence of template or at low template concentrations eventually generated a nonspecific signal, an effect brought about by a primer-dependent artefact (Figure 2B, water control).
To devise a highly sensitive RPA detection system that is not affected by primer artefacts, we developed a probe-based detection method (Figure 3A). The probe we use contains a tetrahydrofuran abasic–site mimic (THF) , flanked in close proximity by nucleotides modified with a fluorophore and a quencher. The fluorescence of the intact construct is low. A block at the 3′-end prevents the oligonucleotide from acting as an amplification primer. Pairing of the probe to complementary DNA enables the recognition of the THF by the double-strand–specific Escherichia coli endonuclease IV (Nfo) . The need for formation of a stable DNA duplex acts as an additional specificity-proofreading step in the context of our detection approach. Subsequent cutting of the probe separates the fluorophore/quencher complex and leads to a measurable increase in fluorescence. The cleavage reaction generates a free 3′ OH-end on the 5′ remnant of the incised probe. This oligomer can then be elongated by Bsu polymerase, thus serving as an amplification primer.
(A) Signal generation by separation of a fluorophore and a quencher depends on cutting of the probe by double-strand specific Nfo.
(B) Arrangement of primers and probes relative to the targets used in Figures 4 and 5. See Protocol S1 for sequences of MRSA isoforms. A PCR fragment that fused an unrelated sequence to the target sites sccIII and orfX served as internal control.
(A) Probe signal of RPA reactions using the primer set orfX/sccIII (see Figure 3). MRSAIII DNA at 10 4 (black, reactions 1–3), 10 3 (red, 4–6), 100 (yellow, 7–9), ten (green, 10–12), or two (purple, 13–17) copies or water (blue, 18–20) served as template.
(B) A plot of the onset time of amplification (defined as passing the 2.5 threshold) in reactions 1 to 12 in Figure 4A against the logarithm of the template copy number reveals a linear relationship.
(A) MRSAI (green), MRSAII (dark blue), MRSAIII DNA (red) at ten copies, or MSSA DNA at 10 4 copies (blue, negative control) or water (yellow, turquoise, negative controls) served as a template (in triplicate for each template condition). See Figure 3 for the arrangement of primers and probes used.
(B) Detection of 50 copies of internal control DNA included in the reactions in (A). Note that two of the negative control sets (blue, yellow) included internal control template, whereas one set of reactions (turquoise) contained water only and served as “double negative” control.
To demonstrate the performance of combined RPA and probe/nuclease-based read-out, we designed primers and probes for the detection of the common hospital pathogen methicillin-resistant Staphylococcus aureus (MRSA Figure 3B) . The sensitivity and reproducibility of RPA were explored by the amplification of the staphylococcal cassette chromosome mec (SCC mec) integration locus of the isoform MRSAIII (Figure 4A, see Protocol S1 for sequences). The onset of amplification at identical starting template quantities of ten, 100, 1,000, or 10,000 copies was virtually simultaneous, demonstrating the robustness of the technique. A plot of the time of beginning amplification against the logarithm of starting copy number reveals a linear relationship (Figure 4B). At low template amounts (two copies), the amplification times are distributed over a wider period, with one reaction failing to generate a signal at all, and the correlation between onset of amplification and template concentration breaks down. However, product detection in samples with such low template concentrations supports the notion that RPA can essentially attain single template copy sensitivity.
The high sensitivity and specificity of RPA allow the design of a multiplex approach in which different amplicons are generated and detected in the same reaction. Three polymorphic alleles of the SCC mec integration region represent the vast majority of known MRSA genotypes (MRSAI through III Figure 5A) . While they share the genomic locus orfX, they differ in the sequence of the SCC mec. The integrating elements of MRSAI and II are homologous to each other except for a 102–base-pair insertion/deletion, while the MRSAIII SCC mec is highly divergent. A primer shared by all alleles was designed for the common region (orfX), whereas primers specific for MRSAI/II and MRSAIII target the different SCC mec variants (sccI/II and III, respectively). To ensure detection of all three isoforms, we designed two highly homologous probes (to account for polymorphisms) for the common region (SATamra1 and SATamra2). Fusing the target sequences for the primers sccIII and orfX to an unrelated DNA sequence created an internal control for the reaction. To verify the activity of each sample, the amplification of this construct was monitored simultaneously with that of the MRSA targets using another probe of appropriate sequence and bearing a different fluorophore/quencher pair (BSFlc). We employed this combination of primers and probes to detected ten genomic copies of the three MRSA types (Figure 5B). By contrast, the closely related methicillin-sensitive Staphylococcus aureus (MSSA 10 4 copies) serving as a negative control did not generate a signal, despite being amplified by suitable primers (see Figure S5). The internal control was concomitantly detected in all samples.
The combination of RPA and the described nuclease-sensitive fluorophore/quencher probes constitutes a DNA amplification/detection system that would require no other equipment than a simple handheld fluorometer, thus making quantitative DNA testing potentially accessible in a nonlaboratory point-of-care environment. We devised an even simpler detection approach by employing lateral-flow dipstick technology. This system is often used as a simple disposable diagnostic device. It employs pairs of specific antibodies to immobilise and detect entities containing two antigenic labels (Figure 6A). Reaction mixtures (see below) are applied to a sample pad soaked in visible gold particles that are coupled to an antibody recognising one antigen. The complexes then travel in a buffer stream through the membrane and an additional, immobilised antibody captures the second antigen. If the antigens are conjoined in a DNA duplex, a coloured line appears at a defined location on the strip. A second line of immobilised antibodies captures the gold-conjugated antibody directly, whether conjoined to the other antigen or not, and serves as a flow control for the strip. In a variation of our probe detection system, we produced such dual antigen complexes by coupling biotin- and carboxyfluorescein (FAM)-bearing oligonucleotides in RPA amplicons (Figure 6B). The 5′-biotinylated primer and its opposing counterpart ensure the efficient amplification of a target for probe binding. The probe, including a 5′-FAM label, an internal THF, and a 3′-blocking group, is incised by Nfo upon binding, creating a 3′ OH substrate for elongation by Bsu. The extension of the probe remnant stabilises its interaction with the biotin-labeled opposing strand and produces an amplicon that contains both antigens, biotin and FAM. The THF/3′ block prevents the production of biotin/FAM-containing primer artefacts, as processing of bona fide duplexes by Nfo adds a critical proofreading step. We used a multiplex approach similar to the one employed in Figure 5 to detect ten copies of each of the three MRSA isoforms and distinguish them from MSSA (Figure 6C). As before, we designed two highly homologous probes (to account for polymorphisms) for the common region (Lfs1 and Lfs2). RPA reactions were followed by the application of the reaction mixtures to the sample pads of lateral-flow strips. Samples containing the MRSA template created biotin/FAM-amplicons and generated visible signals on the antibiotin detection line. In contrast, the MSSA-containing negative control failed to produce a conjoined complex and gave rise to the flow-control line only.
NASBA reactions can be very rapid and produce a large amount of product due to the nature of T7 RNA polymerase, but much of the product will be RNA. Detection is usually accomplished using a Molecular Beacon or similar hybridization probe targeting the single-stranded product, enabling specific detection. NASBA and TMA reactions can be extremely sensitive, and are utilized in a range of clinical diagnostics, including those that screen for infectious diseases in the blood supply.
QPCR and RT-qPCR
Quantitative Endpoint PCR
PCR and RT-PCR are generally used in a qualitative format to evaluate biological samples. However, a wide variety of applications, such as determining viral load, measuring responses to therapeutic agents and characterizing gene expression, would be improved by quantitative determination of target abundance. Theoretically, this should be easy to achieve, given the exponential nature of PCR, because a linear relationship exists between the number of amplification cycles and the logarithm of the number of molecules. In practice, however, amplification efficiency is decreased because of contaminants (inhibitors), competitive reactions, substrate exhaustion, polymerase inactivation and target reannealing. As the number of cycles increases, the amplification efficiency decreases, eventually resulting in a plateau effect.
Normally, quantitative PCR requires that measurements be taken before the plateau phase so that the relationship between the number of cycles and molecules is relatively linear. This point must be determined empirically for different reactions because of the numerous factors that can affect amplification efficiency. Because the measurement is taken prior to the reaction plateau, quantitative PCR uses fewer amplification cycles than basic PCR. This can cause problems in detecting the final product because there is less product to detect.
To monitor amplification efficiency, many applications are designed to include an internal standard in the PCR. One such approach includes a second primer pair that is specific for a &ldquohousekeeping&rdquo gene (i.e., a gene that has constant expression levels among the samples compared) in the reaction (Gaudette and Crain, 1991 Murphy et al. 1990). Amplification of housekeeping genes verifies that the target nucleic acid and reaction components were of acceptable quality but does not account for differences in amplification efficiencies due to differences in product size or primer annealing efficiency between the internal standard and target being quantified.
The concept of competitive PCR&mdasha variation of quantitative PCR&mdashis a response to this limitation. In competitive PCR, a known amount of a control template is added to the reaction. This template is amplified using the same primer pair as the experimental target molecule but yields a distinguishable product (e.g., different size, restriction digest pattern, etc.). The amounts of control and test product are compared after amplification. While these approaches control for the quality of the target nucleic acid, buffer components and primer annealing efficiencies, they have their own limitations (Siebert and Larrick, 1993 McCulloch et al. 1995), including the fact that many depend on final analysis by electrophoresis.
Numerous fluorescent and solid-phase assays exist to measure the amount of amplification product generated in each reaction, but they often fail to discriminate amplified DNA of interest from nonspecific amplification products. Some of these analyses rely on blotting techniques, which introduce another variable due to nucleic acid transfer efficiencies, while other assays were developed to eliminate the need for gel electrophoresis yet provide the requisite specificity. Real-time PCR, which provides the ability to view the results of each amplification cycle, is a popular way of overcoming the need for analysis by electrophoresis.
Quantitative Real-Time PCR
The use of fluorescently labeled oligonucleotide probes or primers or fluorescent DNA-binding dyes to detect and quantitate a PCR product allows quantitative PCR to be performed in real time. Specially designed instruments perform both thermal cycling to amplify the target and fluorescence detection to monitor PCR product accumulation. DNA-binding dyes are easy to use but do not differentiate between specific and nonspecific PCR products and are not conducive to multiplex reactions. Fluorescently labeled nucleic acid probes have the advantage that they react with only specific PCR products, but they can be expensive and difficult to design. Some qPCR technologies employ fluorescently labeled PCR primers instead of probes.
The use of fluorescent DNA-binding dyes is one of the easiest qPCR approaches. The dye is simply added to the reaction, and fluorescence is measured at each PCR cycle. Because fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored as an increase in fluorescent signal. However, preliminary work often must be done to ensure that the PCR conditions yield only specific product. In subsequent reactions, specific amplification can verified by a melt curve analysis. Thermal melt curves are generated by allowing all product to form double-stranded DNA at a lower temperature (approximately 60°C) and slowly ramping the temperature to denaturing levels (approximately 95°C). The product length and sequence affect melting temperature (Tm), so the melt curve is used to characterize amplicon homogeneity. Nonspecific amplification can be identified by broad peaks in the melt curve or peaks with unexpected Tm values. By distinguishing specific and nonspecific amplification products, the melt curve adds a quality control aspect during routine use. The generation of melt curves is not possible with assays that rely on the 5&prime&rarr3&prime exonuclease activity of Taq DNA polymerase, such as the probe-based TaqMan® technology.
Some qPCR strategies employ complementary nucleic acid probes to quantify the DNA target. These probes also can be used to detect single nucleotide polymorphisms (Lee et al. 1993 Bernard et al. 1998). There are several general categories of real-time PCR probes, including hydrolysis, hairpin and simple hybridization probes. These probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but probes can differ in the number and location of the fluorescent reporters.
Hydrolysis probes are labeled with a fluor at the 5&prime-end and a quencher at the 3&prime-end, and because the two reporters are in close proximity, the fluorescent signal is quenched. During the annealing step, the probe hybridizes to the PCR product generated in previous amplification cycles. The resulting probe:target hybrid is a substrate for the 5&prime&rarr3&prime exonuclease activity of the DNA polymerase, which degrades the annealed probe and liberates the fluor (Holland et al. 1991). The fluor is freed from the effects of the energy-absorbing quencher, and the progress of the reaction and accumulation of PCR product is monitored by the resulting increase in fluorescence. With this approach, preliminary experiments must be performed prior to the quantitation experiments to show that the signal generated is proportional to the amount of the desired PCR product and that nonspecific amplification does not occur.
Hairpin probes, also known as molecular beacons, contain inverted repeats separated by a sequence complementary to the target DNA. The repeats anneal to form a hairpin structure, where the fluor at the 5&prime-end and a quencher at the 3&prime-end are in close proximity, resulting in little fluorescent signal. The hairpin probe is designed so that the probe binds preferentially to the target DNA rather than retains the hairpin structure. As the reaction progresses, increasing amounts of the probe anneal to the accumulating PCR product, and as a result, the fluor and quencher become physically separated. The fluor is no longer quenched, and the level of fluorescence increases. One advantage of this technique is that hairpin probes are less likely to mismatch than hydrolysis probes (Tyagi et al. 1998). However, preliminary experiments must be performed to show that the signal is specific for the desired PCR product and that nonspecific amplification does not occur.
The use of simple hybridization probes involves two labeled probes or, alternatively, one labeled probe and a labeled PCR primer. In the first approach, the energy emitted by the fluor on one probe is absorbed by a fluor on the second probe, which hybridizes nearby. In the second approach, the emitted energy is absorbed by a second fluor that is incorporated into the PCR product as part of the primer. Both of these approaches result in increased fluorescence of the energy acceptor and decreased fluorescence of the energy donor. The use of hybridization probes can be simplified even further so that only one labeled probe is required. In this approach, quenching of the fluor by deoxyguanosine is used to bring about a change in fluorescence (Crockett and Wittwer, 2001 Kurata et al. 2001). The labeled probe anneals so that the fluor is in close proximity to G residues within the target sequence, and as probe annealing increases, fluorescence decreases due to deoxyguanosine quenching. With this approach, the location of probe is limited because the probe must hybridize so that the fluorescent dye is very near a G residue. The advantage of simple hybridization probes is their ability to be multiplexed more easily than hydrolysis and hairpin probes through the use of differently colored fluors and probes with different melting temperatures (reviewed in Wittwer et al. 2001).
Some qPCR strategies employ complementary nucleic acid probes to quantify the DNA target. These probes also can be used to detect single nucleotide polymorphisms (Lee et al . 1993 Bernard et al . 1998). There are several general categories of real-time PCR probes, including hydrolysis, hairpin and simple hybridization probes. These probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but probes can differ in the number and location of the fluorescent reporters.
QPCR Products and Resources
GoTaq® qPCR and RT-qPCR kits are available for dye-based or probe-based real-time PCR approaches. GoTaq qPCR Systems contain BRYT Green Dye, which provides maximum amplification efficiency and greater fluorescence than SYBR Green.
GoTaq® Probe Systems are ready-to-use master mixes that simplify reaction assembly for hydrolysis probe-based detection.
Module 1 : Polymerase Chain Reaction
Hello everybody, this is Dr. Vishal Trivedi from department of bioscience and bioengineering IIT Guwahati and today we are going to start a new module and our new module is going to deal with the molecular biology tools. So, the molecular biology tools normally deals with the molecules which are important for the livelihood or for the maintenance of the life. So, there are 4 major molecules which are important for maintaining the life one its DNA, the second is RNA, the third is protein and the fourth is the lipids.
So, molecular biology normally deals with all the macromolecule especially the DNA, RNA and proteins. So, when we talk about a DNA is DNA is been amplified with a technique known as a polymerase chain reactions.
(Refer Slide Time: 01:56)
So, the, as the name suggests, the polymerase chain reaction is a technique which is used to amplify a lot of double-stranded DNA molecules with the same identical size and the sequence by the enzymatic method and the cyclic conditions. What it means is that you have original DNA, which is the original DNA and this original DNA is being amplified for multiple cycles.
And that is how you are actually going to have the multiple copies like you have the 4 multiple copies which are actually going to be identical.
Which means the sequence of the all these fragments are going to be identical to the original sequence and also they are going to be amplified with the help of the enzymes. So, if you want to understand the process of polymerase chain reactions, you have to understand the 2 basic components. One, you have to understand about the DNA. The second you have to understand about the enzymatic machinery, what is important for amplifying the DNA?
So, let us start with understanding the DNA first and then we are going to understand about the machinery and then subsequently we are also going to understand how people have developed the technique and then we are also going to understand all the technical aspects related to polymerase change reactions.
(Refer Slide Time: 03:27)
So, DNA is a nucleic acid and that is composed of the 2 complimentary nucleotide binding blocks chains which means in a typical DNA, you have the 2chains, one is the 5 prime to 3 prime and the 3 prime to 5 prime. Both are these strands are connected by the nucleotides which is present inside and the nucleotide are made up of a phosphate group, 5 carbon sugar and a nitrogen base.
So, as you know that when the base is adding up with the sugar, it is actually forming the nucleoside. And when the nucleoside is being attached with the phosphate, it is called as nucleotide. So the DNA is made up off of the nucleotides. So, it is actually a macro molecule where you have the 1 chain of the nucleotides connected to another chain of nucleotides and these 2 new chains of the nucleotides are complimentary to each other.
(Refer Slide Time: 04:47)
So, DNA has the 4 nitrogen bases, you have the 2 purines like the 2 ringed bases like adenine and guanine. And then you have the 2 pyrimidines like cytosine and thymine. These 2, 4 bases are linked in a repeated pattern by hydrogen bonding between the nitrogen bases. The linking of the 2 complimentary strands is called as the hybridization. So, A is making a pair with T and G is always making a pair with C.
So, what you see is that the A is making a pair with T with the help of the 2 hydrogen bonding whereas the G is making our hydrogen bonding with C with the help of the 3 hydrogen bonding which means, the strength of the A to T interaction is weaker compared to the interaction of G to C.
(Refer Slide Time: 05:46)
And because the A is making a pair with T and G is making a pair with C, the DNA is complimentary in nature. What is mean by complimentary in nature is that if you have a first sequence which is called as the primary strand then you can be able to deduce the complimentary sequence. For example, in this case, we have a primary sequence which is GGC TAT GTG and what you see is that wherever you have the G is actually having a C at a complimentary strands.
So, that is that is the way it is actually going to maintain the complementarity. How the complementarity is going to help you in terms of the you know come conserving the molecules because, if you have the strand 1, you can be able to generate the strand 2, because the strand 1 is complimentary to each other or if you have the strand 2, you can be able to generate a strand 1 and that is the basic principle to which the PCR mechanism is working.
For example, if I have this particular sequence and if I want to synthesize the strand 2 what I can do is I can simply attach a short nucleotide bases and then I can just put T machinery and that machinery is actually going to synthesize the complimentary sequences which means, I am looking for a machinery which actually can recognize this DNA sequence and then that machinery could be able to understand that the DNA is complimentary in nature.
So, what will happen is, if I have this sequence, the machinery is going to sit onto this sequence and then it is actually going to accept the nucleotide from what you are going to supply and then it is actually going to synthesize the complimentary strand. And once it reaches to the end of the
sequence, it will know that now, the synthesis is over. So, it is actually going to terminate. So, now, so far what we have discussed we have discussed about the molecule which is actually going to be amplified during the polymerase chain reactions.
And now, what we are going to understand we are going to understand about the machinery for the DNA synthesis machinery is the basis of the life on the earth or the DNA synthesis machinery is very crucial for duplicating the DNA.
(Refer Slide Time: 08:23)
And DNA synthesis is linked to the cell division as well as the growth. So this is an example of the bacteria. So, what you see is that we have the bacteria which is actually having a single gene genome copy. And what will happen is that the first event itself is that the genome is actually going to duplicate into 2 copies. So, ultimately it is going to have 2 copies of the genome and then it is actually going to be divided by longitudinally and that is how you are actually going to have the 2 bacterial colonies.
Similarly, for in the prokaryote, it has the multiple events through which it actually goes through and then it is actually going to be divided into 2 and that all events are actually being called as the cell cycle. What are these events? You have a G1. So, in the G1, you are actually going to increase the amount of cytosol and you are actually going to prepare the cell for the synthesis phase and then you are actually entering into a synthesis phase.
So, in the synthesis phase or the S phase, you are actually going to do a DNA synthesis which means you are going to make a first copy of DNA and you are going to synthesize the second copy of DNA which means now the DNA is going to be duplicated which means now you are going to have the 2 genomes. Just like in this stage and now it will enter into the G2 phase. G2 is also preparative stage.
So, here also there will be some additional preparative stages. And then the cell will enter into the mitotic phase. And in this phase, the cell is going to divide into 2. So the original cell will again go through with the cell cycle whereas it is actually going to give you the 1 more copy of the cell. For that if you actually block the DNA synthesis, if you block this step, you are actually going to disrupt the cell cycle. And that is how you are actually going to stop the multiplication as well as the growth of particular cell.
(Refer Slide Time: 10:37)
So, process of duplication of the entire genome prior to the cell division. So DNA replication is a process which occurs in the cell prior to the duplication of the cells. And that occurs within the S-phase. So it has a biological significance that the DNA replication is a very accurate process. And it requires to, in order to preserve the integrity of the genome in a successive generation which means if you are actually going to generate the mutations.
Or if you are start generating the modulations within the sequence of the genome, what you got from the original cell, you are eventually going to keep altering the DNA sequences and that
actually is not going to you know, carry the same information because the major function of the DNA is to carry the genetic information. So that it is actually going to allow the daughter cells to keep running the similar kind of metabolism.
So that is why it is important that DNA replication should be accurate. So that it should produce the identical copy in order to preserve the sequence of the DNA as well as it should preserve the integrity of the genome. And that should continue for several generations. In eukaryotes, the replication only occurred during the S phase of the cell cycle. The replication rate in a eukaryote is slower resulting in a higher fidelity accuracy of replication in eukaryotes.
(Refer Slide Time: 12:15)
Now, the DNA replication is having the various characteristic. For example, DNA replication is semi conservative which means the DNA replication is going to be semi conservative means when you are actually going to synthesize for example, if this is the 2 strand 1 and 2, it is actually going to give you the 4 copies of 4 strands. But in this 4 strand, the 1 and 2 are going to be separated and the 3 and 4 are actually going to be synthesized.
So, the new strand, what is been synthesized is actually going to make a pair with the original strands and this process is called as the semi-conservative modes. So this is the 1 and 2 are actually coming from the parents whereas the 3 and 4 are actually the newly synthesized strains. This means the information what you have in the original DNA is going to be divided into 2.
And that is why it is not going to be conservative which means it is not going to make the pair like 1, 2 and 3, 4. It is actually going to make a pair like 1 and 3 and 2 and 4.
That is actually a semi conservative mode of replications. If you want to study more about or if you are interested to study more about how people have discovered the semi-conservative mode, you can easily go through some of the molecular biology book and you can read about the some of the classical experiment what has been designed and how people have discovered that the DNA replication is semi conservative in nature. It starts at the origin of the replication.
For DNA synthesis does not start at random places, it requires a place where it actually can start so that place is called as the origin of replications. In the bacterial cell, you have the single origin of replication whereas in the eukaryotic cell, you have the multiple places of the origin of replications because the bacterial genome is smaller in nature. So, it actually does not require the multiple origin of replications compared to that the eukaryotic genomes are bigger in size.
So they require the multiple origin of replication. So that the application can start at multiple places. And that is how it actually can be able to complete the replications in a given timeframe. Because you know that the replication is important for making a second copy of the genome. So that the cell will enter into the G2 and M phase and that is how it actually going to divide and give you the more number of cells.
So that is why the and as phase is actually the bottleneck of the whole process and that so the DNA replication has to be in synchronous with the mitotic as well as the G2 phase. DNA synthesis by the DNA polymerase always occurs in the direction which is called as the 5 prime to 3 prime directions. So, you know that that DNA is actually a polymer of the nucleotides and nucleotides have 2 strands like 5 prime and 3 prime.
So, if I draw a typical nucleotide structures, what you will see is that this is actually a sugar then this is the base. So, for example, I have put A and here you have the sugar and on this fourth carbon, you have the fifth carbon and then you have the CH2 and then you have the phosphate group. So, this is actually the 5 prime end and this is the 3 prime end because here you have the
OH. No, this is the 3 prime end and this is the 2 prime end. So, this is actually you are going to have OH and that is OH, what you can actually be able to use.
So, if DNA synthesis is occurring, it is actually either utilizing this 5 prime OH or the 3 prime OH which means that is why, the DNA has a directionality which actually goes from 5 prime to 3 prime and 3 prime to 5 prime because this is the complimentary strand. So, the DNA synthesis always occurs in a direction which is called as the 5 prime to 3 prime which means it actually going to start the synthesis on the strand, the strand which is having the 3 prime ends.
And that is how it is actually going to synthesize in this direction because the sentences will be in the direction. It could be unidirectional or bi directional which means it can be either in this direction or it can be in both directions simultaneously. It is a semi discontinuous. For example, so, semi discontinuous means it is actually going to continue for some time and then it is actually going to stop and then it is again going to continue for that and that is why it is actually going to create the 2 different types of strands.
1 is called as the leading strand, the other 1 is called as the lagging strand. So, this is the strand which is called which is actually synthesizing the DNA in the multiple steps is called as the lagging strand whereas the strand which starts from the one end and goes continues like that is called as the leading strand. And for any DNA replication process, you require a primer and that primer is been synthesized by an enzyme which is called as the RNA primer. RNA primers and the primers which are been used in the DNA replication are made up off of the RNA and this has been synthesized by the RNA polymerase.
(Refer Slide Time: 18:01)
DNA replication has 3 steps. It has like initiation. So, in the initiation the DNA is going to be prepared for the synthesis. What is mean by the initiation is that in the initiation step the protein will bind to the DNA. So, you know, the DNA is a double helical structure. So, this double helical structure first has to be straight, so, that you first you have to remove the helicity of this particular DNA. So, you are going to first generate the DNA like double stranded DNA and then you have to actually break the 2 strands separately.
So, that it is actually going to be ready for accepting these this is machinery and that is what the events are happening when they are actually going for the initiation states. In the initiation states, the protein will bind to the DNA and then there will be an opening of double helix DNA. So, that it is actually going to be single stranded DNA. So, the single stranded DNA is going to be generated in the initiation stage then you have the elongation steps.
So, in the elongation steps, the DNA machinery is going to be set on to this and as well as onto this and then it is actually going to start adding the nucleotides and that is how it is actually going to start the synthesis of the second strands. So, in the elongation steps the protein will connect the correct sequence of the nucleotide into continuous new strands of DNA. And once the DNA machinery is going to be reached to the corners like the end of the sequence, it is actually going to stop the synthesis.
And that is what the third step that is third step is the termination which is actually going to stop the DNA synthesis. And once the DNA synthesis is over, the DNA has to again regain to its original form and that is the step what is required if you once you before you do the termination which means once the synthesis is over, the DNA is first going to be converted into a double helical structure and then from the double standard structure, you have to generate a double helical structure.
So, that is why DNA synthesis is a very, fine controlled process where you first have to do the initiation, where you the DNA has to be break into the 2 different strands and then the 2 different strands are going to be you know occupied by the DNA synthesis machinery and what the DNA synthesis machinery is going to do? So, DNA synthesis machinery involves the DNA polymerases which actually going to sit onto the enzyme.
And then they are actually going to read the original sequence and based on the original sequence, they are actually going to add the nucleotides on to the second strand and that is how it is actually going to start the synthesis of the second strand and once they reach to the end, that is what the elongation phase and once they reach to the end of the sequence, they are actually going to start the termination steps. So, once the synthesis is over, it is actually going to generate the double standard DNA and then double standard DNA is actually going to be coiled and then it is going to generate the helical structures.
(Refer Slide Time: 21:20)
There are different enzymes what is involved in this DNA replication process. So, you require the helicases which actually going to separate the 2 different strands. Then you require the primase which is actually our RNA polymerase. So, it is actually going to be required for making a synthesis of the RNA primers. Then you require the single stranded DNA proteins. So, single stranded DNA proteins are actually going to bind to the 2 strands like this, so that they should not come together and again re-anneals.
And that is how it is actually going to maintain the single standard DNA. Then you require the DNA polymerase which is actually going to do a synthesis of new strand and then you required the tethering protein which actually going to stabilize the polymerases onto the strands.
(Refer Slide Time: 22:10)
So, DNA synthesis has the 3 steps. 1 is called as the initiation which is actually going to prepare the DNA for the synthesis. So, what you are doing, you are actually taking a DNA and then you are actually generating the 2 strands then in the elongation steps, we are doing the DNA synthesis. So, once your 2 strands are ready, then what you can do is you are actually going to take and add the primers. So, in this case the RNA primers and so, the both the primers are going to be sit and then you are going to add the enzymes.
So the enzyme is going to be sit on to these strands. And that is how it is actually going to start the synthesis and once the termination, so you can stop the DNA synthesis. So, what will happen it will, once it will reach to this point, that enzyme is actually going to fall from the strands and
that it is actually going to complete the DNA synthesis and it will enter into the terminations. So now what you see is that these are the processes what we do under the DNA synthesis.
And what happens inside the cell can be mimic even outside the cell because all these events can be controlled even by without going through you know some of the enzymes what you require. For example, you require the helicases, so that it actually going to generate the single stranded DNA and that is how it is actually going to make the enough and then you require the single standard DNA binding proteins.
So that the DNA strands what have been generated by the helicases will remain as the single stranded. For these 2 processes can be simply be done by if you heat these particular strands. So if you heat this particular strand, what will happen is that the double standard DNA is going to be open into the single stranded templates. And that is how you can be able to utilize them. Now in the elongations phase, what you can do is you can simply add the small stretches of DNA sequences, which is actually going to be called as the primer.
And that is how it is and then you can add the enzyme. So, that is how you actually going to start the synthesis process. And once it reaches to the end, it is actually going to be a fall from the template and therefore it is actually going to end up into the terminations. So keeping these events and realizing that these events can be done under the invitro conditions. People have conceptualized the process of polymerase chain reaction and that is how they actually started developing the technique. But there are multiple steps and multiple phases in which the polymerase chain reactions is been developed.
(Refer Slide Time: 25:08)
So, there are multiple events what people have done even to develop the PCR for the. So in 1950s people have discovered how the DNA replication is happening and that is being done by the Arthur Kornberg and he discovered the first DNA polymerase and other factors like helicase in and Primers. So, in 1950s when the Arthur Kornberg actually discovered the DNA polymerases as well as the mechanism of DNA replications and helicases and primases, that is how that the people know that the DNA is actually been replicated.
And it requires an enzyme and there is an enzyme. But what is the problem? The problem is the DNA polymerase what has been discovered by Arthur Kornberg is actually the temperature sensitive which means it is cannot withstand a very high temperature. And you know, that, as I said in the in the last slide itself, that when you want to mimic these conditions under the invitro conditions, the only way you can be able to escape the helicases and all other proteins is that you can actually heat up the DNA.
So that the DNA will come into the 2 strands and those 2 strands will remain as a single standard strands only if you keep the temperature very high. But once you bring the temperature high, the enzyme, what has been reported by Dr. Kornberg is actually going to be inactivated. And that is why nothing happened until in 1976 the people have discovered the first thermostable DNA polymerase from the thermo aquaticus and that thermo DNA polymerase is called as the taq DNA polymerase and that taq DNA polymerase is very, stable.
It can be withstand the temperature of 95 degrees Celsius which means you can be able to use this enzyme on multiple rounds. And you do not need to, you know, you do not need to add the enzyme on, you know, for every cycle you have to continue. Then in 1983, Kary mullis synthesizes that DNA oligo probes for sickle cell anemia. And then 1983, he only did the repeated thermal cycling was first used to clone a small segment of genome DNA.
And then in 1984, the Kary mullis and Tom white tried the design experiment to test PCR on the genomic DNA but the amplified product was not visible in the agarose. Then in 1985, the first patent was filed for its application regarding the detection of the sickle cell anemia mutations. And the 1985, the first use of thermostable DNA polymerase in PCR was started out of only 2 enzymes, the taq DNA polymerase, known at the time the taq was found more suitable for the PCR.
And 1985 to 1987 people have discovered a lot about the different types of instruments and so there are a lot of discoveries and development later on with the help of the instrumentations.
(Refer Slide Time: 28:29)
So what basically people are doing in a polymerase chain reaction. So PCR is a repeated cycling reaction that involves the mechanism of DNA replications. It results in the production of multiple copies of DNA from a single 1, the whole process involves 3 events, one is called denaturation, annealing, and the elongations. So what happens so as you can imagine that you started with original copy of the double stranded DNA.
So under the denaturation, when you have heated the sample at 95 degree Celsius, what will happen is that the 2 strands of the original copy are going to be separated and it will give you the 2 strands. Now what you are going to do is you are going to enter into the annealing phase where you are actually going to lower down the temperature. So, that the primers are going to buy and then you are actually going to have the primers.
So it is actually going to add the primers and that is how it is actually going to attach the primer on 1 end of this DNA as well as the 1 end of this DNA. And then the enzyme will enter into the elongation phase. And the enzyme is actually going to utilize this small primer DNA and that is how it is actually going to synthesize the whole strand and that is how you are actually going to get the double stranded DNA at the end.
So, this is actually is going to constitute the first cycle or the first cycle. After the first cycle, since you started with the 1 DNA, you are actually going to get 2 DNA. But as you can see, it is actually a semi-conservative, which means the 1 strand is from the original DNA. And the second strand is the newly synthesized DNA strand. Now, you can imagine that the same thing happened in the second cycle.
But now, this one also is these 2 strands are also going to serve as a template. And that is how you are actually going to get the 4 copies and in the third cycle, you are going to get the 8 copies which means a DNA fragment of interest is used as a template from which a pair of primer or short oligonucleotide complimentary to both the double strand of the DNA are made to prime the DNA synthesis where the direction of synthesis or the extension is 5 prime to 3 prime as it was in the case of DNA replications the number of amplified DNA.
Or the amplicons increases exponentially per cycle for example, if you start with the 1 DNA molecule, after first cycle, it is going to be to DNA, after second cycle it is going to be 4, after the third cycle it is going to be 8. So, if you continue that it is actually continuing like this cycle like so. So, it is actually doubling after every cycle and that is how it is actually going to give you very high amplification even from the 1 molecule.
So, if you imagine that if I started with 1 microgram of DNA, it is actually going to be several microgram of DNA and even within that 20, 25 cycles. So, the amount of amplified DNA you can be able to calculate simply by putting this formula that is the C is equal to C0 1 plus E to the power n where the C is the final amount of DNA, C0 is the initial amount of DNA, E is the efficiency and n is the number of cycles and S is the slope of the exponential phase and that you can be able to calculate from the this particular equations.
So, you can see that the polymerase chain reaction actually utilizes the similar concept what is happening in the during the DNA replications but instead of utilizing the multiple factors and such complicated mechanisms, what you are doing is you are simply heating the reactions, you are making the 2 strands, then you are adding the primers, which means the small DNA stretches and then you are asking to enzyme to utilize these primers to synthesize and then once the synthesis is over, then again you are bringing the temperature back and continuing the same cycle and that is how you are actually making the multiple reactions.
(Refer Slide Time: 32:58)
So the polymerase chain reaction requires the 4 events. So if you set up the polymerase chain reactions, you require the initial denaturation which means you have to heat the PCR mixture at 94 to 95th degree for 10 minutes to completely denature the template DNA. So when you are going to start the PCR reactions, you have to first bring the reactions and then you are going to
first do the initial denaturation where you are actually going to heat up the mixture at 95 degree Celsius for 5 to 10 minutes.
And then you are actually going to enter into the stage 2 where you are going to again heat up the template for 30 seconds to 45 seconds. And that actually is going to be our denaturation steps. So in the denaturation steps, this is the first step in which the double stranded DNA is going to be denature to form the 2 single standard DNA by heating the DNA at 95 degrees Celsius for 15 to 30 seconds. Now, you are going to lower down the temperature.
So that is going to be annealing steps. So in the annealing step, this is the annealing step where the lower temperature like 50 to 65 degree Celsius, the primers are allowed to bind to the template DNA and link time is 15 to 30 seconds and it depends on the length and the bases of the primers. So what you are going to do is you are you have lower down the temperature so that the primers which are actually been floating and which are small stretch of DNA are now go and bind to its complimentary DNA present onto the template.
And now, the enzyme will recognize this particular D complex and that is how the enzyme will start the synthesis. So after the annealing it will enter into the elongation phase. So this is the synthesis step where the polymerase chain performed the synthesis of the new strand in the 5 prime to 3 prime directions using the primers the dNTPs or the deoxyribo nucleotide triphosphates.
And average DNA polymerase adds about 1000 base pair per minute which means, after the annealing is over and the primer dimers has formed the complex, you are again going to increase the temperature. So that you will ensure that the template DNA should not bind to each other and then the enzyme will come sit onto these complexes and it is actually going to do a DNA synthesis.
And as an average 1000 base pair is going to be synthesized within the minute where if you supply the adequate amount of the deoxyribo nucleotides in the mixture. So, step 1 and 2, 3 will make 1 cycle. So this stage through means like step 1, 2 and 3 is actually constituting the first 1
cycle. And you can ask the machine to continue the cycle for another
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Molecular Analysis of Proteins
In many cases it may not be desirable or possible to study DNA or RNA directly. Proteins can provide species-specific information for identification as well as important information about how and whether a cell or tissue is responding to the presence of a pathogenic microorganism. Various proteins require different methods for isolation and characterization.
Polyacrylamide Gel Electrophoresis
A variation of gel electrophoresis, called polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins. In PAGE, the gel matrix is finer and composed of polyacrylamide instead of agarose. Additionally, PAGE is typically performed using a vertical gel apparatus (Figure 6). Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on their net charges. Alternatively, proteins can be denatured and coated with a negatively charged detergent called sodium dodecyl sulfate (SDS), masking the native charges and allowing separation based on size only. PAGE can be further modified to separate proteins based on two characteristics, such as their charges at various pHs as well as their size, through the use of two-dimensional PAGE. In any of these cases, following electrophoresis, proteins are visualized through staining, commonly with either Coomassie blue or a silver stain.
Figure 6. Click for a larger image. (a) SDS is a detergent that denatures proteins and masks their native charges, making them uniformly negatively charged. (b) The process of SDS-PAGE is illustrated in these steps. (c) A photograph of an SDS-PAGE gel shows Coomassie stained bands where proteins of different size have migrated along the gel in response to the applied voltage. A size standard lane is visible on the right side of the gel. (credit b: modification of work by “GeneEd”/YouTube)
Think about It
Clinical Focus: Karni, Part 3
This example continues Karni’s story that started in Microbes and the Tools of Genetic Engineering and above.
Figure 7. A bulls-eye rash is one of the common symptoms of Lyme diseases, but up to 30% of infected individuals never develop a rash. (credit: Centers for Disease Control and Prevention)
When Karni described her symptoms, her physician at first suspected bacterial meningitis, which is consistent with her headaches and stiff neck. However, she soon ruled this out as a possibility because meningitis typically progresses more quickly than what Karni was experiencing. Many of her symptoms still paralleled those of amyotrophic lateral sclerosis (ALS) and systemic lupus erythematosus (SLE), and the physician also considered Lyme disease a possibility given how much time Karni spends in the woods. Karni did not recall any recent tick bites (the typical means by which Lyme disease is transmitted) and she did not have the typical bull’s-eye rash associated with Lyme disease (Figure 7). However, 20–30% of patients with Lyme disease never develop this rash, so the physician did not want to rule it out.
Karni’s doctor ordered an MRI of her brain, a complete blood count to test for anemia, blood tests assessing liver and kidney function, and additional tests to confirm or rule out SLE or Lyme disease. Her test results were inconsistent with both SLE and ALS, and the result of the test looking for Lyme disease antibodies was “equivocal,” meaning inconclusive. Having ruled out ALS and SLE, Karni’s doctor decided to run additional tests for Lyme disease.
- Why would Karni’s doctor still suspect Lyme disease even if the test results did not detect Lyme antibodies in the blood?
- What type of molecular test might be used for the detection of blood antibodies to Lyme disease?
We’ll return to Karni’s example in later pages.
Reagents and Solutions
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX Unavailable for suppliers, see APPENDIX Unavailable.
For a discussion of how to select enhancer agents, see 2.
25% acetamide (20 µl/reaction 5% final)
5 M N,N,N-trimethylglycine (betaine 20 µl/reaction 1 M final)
40% polyethylene glycol (PEG) 8000 (20 µl/reaction 8% final)
Glycerol (concentrated 10 µl/reaction 10% final)
Dimethylsulfoxide (DMSO concentrated 5 µl/reaction 5% final)
Formamide (concentrated 5 µl/reaction 5% final)
1 U/µl Perfect Match Polymerase Enhancer (Strategene 1 µl/reaction 1 U final)
10 mg/ml acetylated bovine serum albumin (BSA) or gelatin (1 µl/reaction 10 µg/ml final)
1 to 5 U/µl thermostable pyrophosphatase (PPase Roche Diagnostics 1 µl/reaction 1 to 5 U final)
5 M tetramethylammonium chloride (TMAC betaine hydrochloride 1 µl/reaction 50 mM final)
0.5 mg/ml E. coli single-stranded DNA-binding protein (SSB Sigma 1 µl/reaction 5 µg/ml final)
0.5 mg/ml Gene 32 protein (Amersham Pharmacia Biotech 1 µl/reaction 5 µg/ml final)
10% Tween 20, Triton X-100, or Nonidet P-40 (1 µl/reaction 0.1% final)
1 M (NH4)2SO4 (1 µl/reaction 10 mM final use with thermostable DNA polymerases other than Taq)
MgCl2-free PCR buffer, 10×
Store indefinitely at −20°C
This buffer can be obtained from Promega it is supplied with Taq DNA polymerase.
For 2 mM 4dNTP mix: Prepare 2 mM each dNTP in TE buffer, pH 7.5 ( APPENDIX Unavailable). Store up to 1 year at −20°C in 1-ml aliquots.
For 25 mM 4dNTP mix: Combine equal volumes of 100 mM dNTPs (Promega). Store indefinitely at −20°C in 1-ml aliquots.
Amplification technique for proteins similar to PCR for DNA? - Biology
Publisher: Horizon Bioscience
Editors: Vadim V. Demidov and Natalia E. Broude Boston University, USA
Publication date: July 2004
Pages: xii + 336
DNA amplification is the cornerstone of modern biotechnology and it is also a key procedure in numerous basic studies involving DNA and other biomolecules. Polymerase chain reaction (PCR) is still the most popular amplification method, however alternatives to PCR have successfully invaded the area. The emergence of such methodologies has significantly widened the range of approaches for DNA amplification and dramatically improved the technological abilities of basic and applied researchers in various fields of life sciences. Whereas most books on DNA amplification focus on PCR-based technologies, this volume presents a wider range of methods to amplify DNA with an emphasis on their diverse applications. The book covers both well-established and newly-developed protocols including ligation-based thermocycling approaches, real-time PCR and other new PCR developments, plus several powerful non-PCR isothermal DNA amplification techniques, for example: real-time strand displacement amplification (SDA), rolling-circle amplification (RCA) and multiple-displacement amplification (MDA). An entire section is devoted to a group of enzymes, both natural and engineered, which are employed for DNA amplification and related purposes. In addition, the use of DNA amplification in the detection of non-DNA analytes is presented. Written and edited by leading experts in the field, this book serves as a practical tool and an invaluable reference source for a broad audience of academic researchers and industry biotechnologists who use DNA amplification techniques.
&bull Covers theory, practice and applications
&bull Contains well-established and newly-developed protocols
&bull Comprehensive and up-to-date
&bull Includes the latest technology
&bull A practical laboratory manual
&bull Aimed at a broad audience
&bull Fully illustrated throughout
&bull Useful index
"I . find it extremely fascinating. I will certainly use it in one of my biophysics/biochemistry courses." from Bengt Nolting, Prussian Private Institute of Technology, Berlin .
"Must be a great book! I will buy it as soon as it becomes available. Good book is worth any money. " from Igor Kutyavin, Research Fellow, Epoch Pharmaceuticals Inc .
"Nice book. I want to have it!" from Juan Aymami, University of Catalunya, Spain .
"Recommended." by Microbiology Today (2004) 31: 201 .
". the book should be an essential tool and reference source to researchers in both academia and industry." from Joshua Marcy in Drug Discovery and Development (2004) 7: 92.
DNA Amplification is a compendium of articles edited by Vadim Demidov and Natalia Broude, including two articles by the editors. The book stands out from the field of similar offerings by virtue of including chapters covering novel and cutting-edge PCR-based methods along with up and coming non-PCR amplification techniques. Readers looking for ideas on how to address research questions where more 'traditional' approaches have not proven quite satisfactory will find this an excellent and informative survey of new thinking. Moreover, explicit methods sections are provided by most authors so readers can readily implement ideas generated by the book. Overall, the book is an engaging and worthwhile read." from David J. Lane Ph.D., Vice President R and D, Hamilton Thorne Biosciences .
"An essential book for all people in this field." from Sajjad Sarikhan, Zanjan University, Iran .
". a practical tool and invaluable reference source of latest DNA amplification technologies for academic and industrial scientists . " from Expert Rev. Mol. Diagn. (2005) 5: 127-129 .
"This book . is directed at those with a solid background in molecular biology who desire knowledge of cutting-edge applications . a good addition to the library of researchers in molecular biology." from Emerg. Infect. Dis. (2005) 11: (2) .
"comprehensive and didactical content" Monica Ramirez Concepto Azul, ECUADOR .
Section 1. Enzymes Used in DNA Amplification
Thermostable Chimeric DNA Polymerases with High Resistance to Inhibitors
Andrey R. Pavlov, Nadejda V. Pavlova, Sergei A. Kozyavkin and Alexei I. Slesarev
Phi29 DNA Polymerase, a Potent Amplification Enzyme
Margarita Salas, Miguel de Vega, José M. Lázaro and Luis Blanco
High-Fidelity Thermostable DNA Ligases as a Tool for DNA Amplification
Section 2. Thermocycling Methods of DNA Amplification
High Multiplexity PCR Based on PCR Suppression
Natalia E. Broude, Adriaan W. van Heusden and Richard Finkers
On-Chip PCR: DNA Amplification and Analysis on Oligonucleotide Microarrays
Martin Huber, Christian Harwanegg, Manfred W. Mueller and Wolfgang M. Schmidt
Analysis of Somatic Mutations via Long-Distance Single Molecule PCR
Yevgenya Kraytsberg, Ekaterina Nekhaeva, Connie Chang, Konstantin Ebralidse and Konstantin Khrapko
Digital PCR Analysis of Allelic Status in Clinical Specimens
Wei Zhou, Tanisha Williams, Cecile Colpaert, Aki Morikawa and Diansheng Zhong
Application of Real-Time Quantitative PCR in the Analysis of Gene Expression
Manohar R. Furtado, Olga V. Petrauskene and Kenneth J. Livak
Quantitative Genetic Analysis with Multiplex Ligation-Dependent Probe amplification (MLPA)
A.O.H Nygren, A. Errami and J.P. Schouten
Section 3. Isothermal Methods of DNA Amplification
Homogeneous Real-Time Strand Displacement amplification
David M. Wolfe, Sha-Sha Wang, Keith Thornton, Andrew M. Kuhn, James G. Nadeau and Tobin J. Hellyer
Loop-Mediated Isothermal Amplification (LAMP) of DNA Analytes
Tsugunori Notomi, Kentaro Nagamine, Yasuyoshi Mori and Hidetoshi Kanda
Ligation-Mediated Rolling Circle DNA Amplification for Non-Gel Detection of Single Nucleotide Polymorphisms (SNPs)
Rolling-Circle Amplification of Duplex DNA Sequences Assisted by PNA Openers
Heiko Kuhn and Vadim V. Demidov
Phi29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing
John C. Detter, John R. Nelson and Paul M. Richardson
Multiple-Displacement Amplification (MDA) of Whole Human Genomes from Various Samples
Roger S. Lasken, Seiyu Hosono and Michael Egholm
Section 4. DNA Amplification in Detection of Non-DNA Analytes
Enhanced Protein Detection Using Real-Time Immuno-PCR
Michael Adler and Christof M. Niemeyer
Rolling Circle Amplification in Multiplex Immunoassays
Michael C. Mullenix, Richard S. Dondero, Hirock D. Datta, Michael Egholm, Stephen F. Kingsmore and Lorah T. Perlee
DNA amplification is the cornerstone of modern biotechnology and it is also a key procedure in numerous basic studies involving DNA molecules. All methods for DNA amplification have rested on the concept of DNA strand complementarity discovered by James Watson and Francis Crick fifty years ago. To an equal extent, these methods became possible with the discovery of DNA polymerases first identified by Arthur Kornberg soon after the Watson-Crick discovery and DNA ligases discovered in 1967 by Martin Gellert, Charles Richardson, Jerard Hurwitz, Robert Lehman and others. Using these enzymes (and later their thermostable variants), a variety of isothermal and temperature-cycling amplification techniques have been developed starting in late 1980s. Among these techniques, Kari Mullis' polymerase chain reaction (PCR) was the first one and it is still the most popular amplification method. Yet, some alternatives to PCR have also successfully invaded the area. The emergence of such methodologies significantly widened the range of approaches for DNA amplification and dramatically changed the abilities of basic and applied researchers in various fields of life sciences. It will not be an exaggeration to say that now no research related to DNA can be performed without the employment of DNA amplification procedures.
Despite the importance of this topic we found to our surprise that only a few books were published that deal with the subject. Moreover, these books cover mostly PCR-based techniques and/or describe the use of PCR and other DNA amplification approaches for specific goals, such as clinical analysis, environmental microbiology, forensics, etc. This information shortage was a major motivation for us to compile a book on a wider range of methods for DNA amplification with emphasis on their diverse applications. Besides, almost twenty years after PCR was invented, we now are witnessing a new stage in the craft of DNA amplification thanks to the introduction of real-time PCR, several powerful non-PCR DNA amplification techniques and microarray technologies. In an attempt to represent the current state-of-the-art our book covers both well-established and newly-developed protocols with promising potential.
Although the book goes far beyond PCR by presenting a number of isothermal assays along with the ligation-based thermocycling approaches, PCR remains, despite some limitations, the dominant diagnostic technique for target DNA amplification and analysis, and recently this primary method has been systematically improved in many ways. That is why a significant part of our book is devoted to new PCR developments. A separate section is devoted to a group of enzymes, both natural and engineered, which are employed for DNA amplification and related purposes. We also present here the use of DNA amplification in the detection of non-DNA analytes. Note that we do not consider per se the methods for the detection of amplicons obtained by one way or another except for those few that establish a new potent amplification approach, as in the case of real-time PCR or real-time strand displacement amplification (SDA).
We hope that our book will serve as a practical tool and reference source for a broad audience of academic researchers and industry biotechnologists who rely in their work on DNA amplification techniques. We are very grateful to all the contributors and to the publisher who made this book possible.
Vadim V. Demidov and Natalia E. Broude