Cleaved Amplified Polymorphic Sequences (CAPS)

Cleaved Amplified Polymorphic Sequences (CAPS) polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus-specific oligonucleotide primers.

How It Works

The CAPS assay uses amplified DNA fragments that are digested with a restriction endonuclease to display RFLP.

Unique sequence primers are used to amplify a mapped DNA sequence from two related individuals (for example, from two different inbred ecotypes), A/A and B/B, and from the heterozygote A/B. The amplified fragments from A/A and B/B contain two and three RE recognition sites, respectively. In the case of the heterozygote A/B, two different PCR products will be obtained, one which is cleaved three times and one which is cleaved twice. When fractionated by agarose or acrylamide gel electrophoresis, the PCR products digested by the RE will give readily distinguishable patterns. Some bands will appear as doublets.

Advantages

• Most CAPS markers are co-dominant and locus-specific.
• Most CAPS genotypes are easily scored and interpreted.
• CAPS markers are easily shared between laboratories.
• CAPS assay does not require the use of radioactive isotopes, and it is more amenable, therefore, to analyses in clinical settings.

Developing CAPS markers

• Sequence the RFLP probe.
• Design primers to amplify 800–2,000-bp DNA fragments. Targeting introns or 3' untranslated regions should increase the chance of finding polymorphisms
• The PCR product is cloned and sequenced.
• PCR amplify DNA fragments from target genotypes, separately digest the amplicons with one or more restriction emzymes.
• Screen the digested amplicons for polymorphism on gels stained with ethidium bromide. 

The Derived Cleaved Amplified Polymorphic Sequences (dCAPS) assay is a modification of CAPS (or alternatively, PCR-RFLP) technique for detection of Single Nucleotide Polymorphisms (SNPs). In dCAPS assay a mismatches in PCR primer are used to create restriction endonuclease (RE)-sensitive polymorphism based on the target mutation. This technique is useful for genotyping known mutations and genetic mapping of isolated DNAs.

Similar to the CAPS technique, this method is simple, relatively inexpensive, and uses the ubiquitous technologies of PCR, restriction digestion and standard agarose gel electrophoresis.

How It Works

The dCAPS technique introduces or destroys a restriction enzyme recognition sites by using primers that containing one or more micmatches to the template DNA. The PCR product modified in this manner is then subjected to restriction enzyme digestion and the presence or absence of the SNP is determined by the resulting restriction pattern.

Example

Applications of dCAPS primers

1.        To create a restriciton site that is dependent on the presence or absence of the SNP allele in question

2.        To introduce a specific restriction site for each of two alleles being analyzed, to positively identify homozygotes for a particular allele without the possibbility of mis-scoring due to partial restriciton enzyme digestion

3.        To disrupt an additional restriction site situated in close proximity to the CAPS polymorphism to be analyzed

Amplified Fragment Length Polymorphism (AFLP)

Amplified Fragment Length Polymorphisms (AFLPs) are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites.

The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA.

After final amplification, selectively amplified fragments are separated by gel electrophoresis and visualized autoradiographically. MseI-MseI fragments are excluded from the autorad because only EcoRI-directed primers are normally labeled. Typically, the autorad has 100-300 fingerprints with sizes ranging from 80 to 500 nucleotides. Only a subset (10-40) of these total bands is polymorphic between two related individuals, such as Arabidopsis thaliana Columbia and Landsberg erecta ecotypes.

Using 3-bp selective primer extensions gives 128 possible linker combinations. Therefore, 128 subsets of genomic DNA can be readily amplified. Thus, thousands of markers can be generated quite rapidly.

Weaknesses of AFLP

l        Proprietary technology is needed to score heterozygotes and ++ homozygotes. Otherwise, AFLP must be dominantly scored.

l        Developing locus-specific markers from individual fragments can be difficult.

l        Need to use different kits adapted to the size of the genome being analyzed.

Restriction Fragment Length Polymorphism (RFLP)

Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination.

Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific.

An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.

The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.).

How It Works

SNPs or INDELs can create or abolish restriction endonuclease (RE) recognition sites, thus affecting quantities and length of DNA fragments resulting from RE digestion.

Genotyping

Developing RFLP probes

Total DNA is digested with a methylation-sensitive enzyme (for example, PstI), thereby enriching the library for single- or low-copy expressed sequences (PstI clones are based on the suggestion that expressed genes are not methylated).

The digested DNA is size-fractionated on a preparative agarose gel, and fragments ranging from 500 to 2000 bp are excised, eluted and cloned into a plasmid vector (for example, pUC18).

Digests of the plasmids are screened to check for inserts.

Southern blots of the inserts can be probed with total sheared DNA to select clones that hybridize to single- and low-copy sequences.

The probes are screened for RFLPs using genomic DNA of different genotypes digested with restriction endonucleases. Typically, in species with moderate to high polymorphism rates, two to four restriction endonucleases are used such as EcoRI

, EcoRV, and HindIII. In species with low polymorphism rates, additional restriction endonucleases can be tested to increase the chance of finding polymorphism.

PCR-RFLP

Isolation of sufficient DNA for RFLP analysis is time consuming and labor intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time. An alternative name for the technique is Cleaved Amplified Polymorphic Sequence (CAPS) assay.

Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.

How It Works

Unlike traditional PCR analysis, RAPD (pronounced "rapid") does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel.

Example

RAPD is an inexpensive yet powerful typing method for many bacterial species.

Silver-stained polyacrylamide gel showing three distinct RAPD profiles generated by primer OPE15 for Haemophilus ducreyi isolates from Tanzania, Senegal, Thailand, Europe, and North America.

Selecting the right sequence for the primer is very important because different sequences will produce different band patterns and possibly allow for a more specific recognition of individual strains.

Limitations of RAPD

Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.

PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible.

Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

Developing Locus-specific, Co-Dominant Markers from RAPDs

The polymorphic RAPD marker band is isolated from the gel.

It is amplified in the PCR reaction.

The PCR product is cloned and sequenced.

New longer and specific primers are designed for the DNA sequence, which is called the Sequenced Characterized Amplified Region Marker (SCAR).

Real-Time qRT-PCR

Real-Time qRT-PCR (Real Time quantitative Reverse Transcription PCR) is a major development of PCR technology that enables reliable detection and measurement of products generated during each cycle of PCR process. This technique became possible after introduction of an oligonucleotide probe, which was designed to hybridize within the target sequence. Cleavage of the probe during PCR because of the 5' nuclease activity of Taq polymerase can be used to detect amplification of the target-specific product.

How It Works

The following techniques can be used to monitor degradation of the probe: intercalation of double-stranded DNA-binding dyes 32P probe labeling labeling of the probe with fluorescent dyes TaqMan assay (named after Taq DNA polymerase) was one of the earliest methods introduced for real time PCR reaction monitoring and has been widely adopted for both the quantification of mRNAs and for detecting variation. The method exploits the 5' endonuclease activity of Taq DNA polymerase to cleave an oligonucleotide probe during PCR, thereby generating a detectable signal. The probes are fluorescently labeled at their 5' end and are non-extendable at their 3' end by chemical modification. Specificity is conferred at three levels: via two PCR primers and the probe. Applied Biosystems probes also include a minor groove binder for added specificity. Applications of real time quantitative RT-PCR: relative and absolute quantification of gene expression, validation of DNA microarray results, variation analysis, counting bacterial, viral, or fungal loads, etc.

Nomenclature commonly used in real time quantitative RT-PCR: Baseline is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument. ΔRn is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number. Threshold is an arbitrary level of fluorescence chosen on the basis of the baseline variability. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots. Ct is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold. The Ct is a basic principle of real time PCR and is an essential component in producing accurate and reproducible data.

Polymerase Chain Reaction (PCR) Illustrations

Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. The method involves using short DNA sequences called primers to select the portion of the genome to be amplified. The temperature of the sample is repeatedly raised and lowered to help a DNA replication enzyme copy the target DNA sequence. The technique can produce a billion copies of the target sequence in just a few hours.

Illustrations

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Polymerase Chain Reaction (PCR)

What is PCR?

Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and inexpensive technique used to "amplify" - copy - small segments of DNA. Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification.

Often heralded as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993.

What is PCR used for?

Once amplified, the DNA produced by PCR can be used in many different laboratory procedures. For example, most mapping techniques in the Human Genome Project (HGP) rely on PCR.

PCR is also valuable in a number of newly emerging laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders.

How does PCR work?

To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes - builds - two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.

The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.

The Nobel Prize in Chemistry 1993

Kary B. Mullis, Michael Smith

The Nobel Prize in Chemistry 1993

Nobel Prize Award Ceremony

Kary B. Mullis

Michael Smith

Kary B. Mullis

Michael Smith

The Nobel Prize in Chemistry 1993 was awarded "for contributions to the developments of methods within DNA-based chemistry" jointly with one half to Kary B. Mullis "for his invention of the polymerase chain reaction (PCR) method"and with one half to Michael Smith "for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies".

Photos: Copyright © The Nobel Foundation

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What is RT PCR?

RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA). The technique consists of two parts:

• The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and
  
• The amplification of a specific cDNA by the polymerase chain reaction (PCR).

RT-PCR has been used to measure viral load with HIV and may also be used with other RNA viruses such as measles and mumps.

How was PCR (polymerase chain reaction) discovered?

PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 128 one night on his motorcycle. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his motorcycle trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in chemistry with Michael Smith in 1993.
As Mullis has written in the Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."

What is the purpose of doing a PCR (polymerase chain reaction)?

To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses -- to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of an Egyptian mummy, establish paternity or biological relationships, etc.. Accordingly, PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material.

How is PCR (polymerase chain reaction) done?

As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step -- denatauration (alteration of structure), annealing (joining), and extension -- takes place at a different temperature:

1. Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.
   
2. Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
   
3. Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.

With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

What is PCR (polymerase chain reaction)?

PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient so that untold numbers of copies can be made of the DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA:

• Two "primers", short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;
  
• An enzyme called polymerase that moves along the segment of DNA, reading its code and assembling a copy; and
  
• A pile of DNA building blocks that the polymerase needs to make that copy. 

NCBI: PCR

Introduction

PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).

How It Works

The PCR reaction requires the following components: DNA template - the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other. DNA polymerase - a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The first and most commonly used of these enzymes is Taq DNA polymerase (from Thermis aquaticus), whereas Pfu DNA polymerase (from Pyrococcus furiosus) is used widely because of its higher fidelity when copying DNA. Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: 1) they can generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant. Primers - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer. Nucleotides (dNTPs or deoxynucleotide triphosphates) - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands. RT-PCR (Reverse Transcription PCR) is PCR preceded with conversion of sample RNA into cDNA with enzyme reverse transcriptase . Applications of PCR: cloning, genetic engineering, sequencing Limitations of PCR and RT-PCR: The PCR reaction starts to generate copies of the target sequence exponentially. Only during the exponential phase of the PCR reaction is it possible to extrapolate back to determine the starting quantity of the target sequence contained in the sample. Because of inhibitors of the polymerase reaction found in the sample, reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify target sequence at an exponential rate and a "plateau effect" occurs, making the end point quantification of PCR products unreliable. This is the attribute of PCR that makes Real-Time Quantitative RT-PCR so necessary.

The Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying small quantities of DNA. The development of this technique resulted in an explosion of new techniques in molecular biology (and a Nobel Prize for Kary Mullins in 1993) as more and more applications of the method were published. The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus auquaticus that was discovered in hot springs. This DNA polymerase is stable at the high temperatures need to perform the amplification, whereas other DNA polymerases become denatured.
Since this technique involves amplification of DNA, the most obvious application of the method is in the detection of minuscule amounts of specific DNAs. This important in the detection of low level bacterial infections or rapid changes in transcription at the single cell level, as well as the detection of a specific individual's DNA in forensic science (like in the O.J. trial). It can also be used in DNA sequencing, screening for genetic disorders, site specific mutation of DNA, or cloning or subcloning of cDNAs.

The Reaction


PCR, like DNA sequencing, is based on the DNA polymerization reaction. A primer and dNTPs are added along with a DNA template and the DNA polymerase (in this case, Taq). The main difference with PCR is that, in addition to using a primer that sits on the 5' end of the gene and makes a new strand in that direction, a primer is made to the opposite strand to go in the other direction. The original template is melted (at 94^oC), the primers anneal (@ 45-55^oC) and the polymerase makes two new strands (@ 72^oC), doubling the amount of DNA present. This provides 2 new templates for the next cycle. The DNA is again melted, primers anneal, and the Taq makes 4 new strands:
Click here to download a short movie of the PCR reaction.
OR . . .
Click here to download a static figure of the PCR reaction (if the movie doesn't work with your web browser).


Figure 1
Notice:

• Every cycle results in a doubling of the number of strands DNA present.
• After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers.

The result is a dramatic amplification of a the DNA that exists between the primers. These cycles are repeated 20 to 40 times, each cycle providing 2 new templates for the next cycle. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 X 10¹². The reaction is performed in a thermocycler, which is programmable heating block that will cycle between melting, annealing and polymerization temperatures.

Limitations/Difficulties


While a very powerful technique, PCR can also be very tricky. The polymerase reaction is very sensitive to the levels of divalent cations (especially Mg²⁺) and nucleotides, and the conditions for each particular application must be worked out. Primer design is extremely important for effective amplification. The primers for the reaction must be very specific for the template to be amplified. Cross reactivity with non-target DNA sequences results in non-specific amplification of DNA. Also, the primers must not be capable of annealing to themselves or each other, as this will result in the very efficient amplification of short nonsense DNAs. The reaction is limited in the size of the DNAs to be amplified (i.e., the distance apart that the primers can be placed). The most efficient amplification is in the 300 - 1000 bp range, however amplification of products up to 4 Kb has been reported. Also, Taq polymerase has been reported to make frequent mismatch mistakes when incorperating new bases into a strand.
The most important consideration in PCR is contamination. If the sample that is being tested has even the smallest contamination with DNA from the target, the reaction could amplify this DNA and report a falsely positive identification. For example, if a technician in a crime lab set up a test reaction (with blood from the crime scene) after setting up a positive control reaction (with blood from the suspect) cross contamination between the samples could result in an erroneous incrimination, even if the technician changed pipette tips between samples. A few blood cells could volitilize in the pipette, stick to the plastic of the pipette, and then get ejected into the test sample. The powerful amplification of PCR may be able to detect this cross contamination of samples. Modern labs take account of this fact and devote tremendous effort to avoiding this problem.

Procedure:


Primers
As stated above, the selection of primers is very important to the efficiency of the reaction. Usually the primers are custom synthesized based on the sequence of the DNA that is being amplified. In your reactions, two primers would have to be made for each of the inserts and the primers that you use would be based on which insert you have in your plasmid. However, since all of the inserts are in the pBluescript plasmid, we can take advantage of the vector sequences that are common to all of the plasmids. For this reason you will all be using the same primers; one primer from the vector sequences at the 5' end of your insert and one from vector sequences at the 3' end of your insert. When the products are run on agarose gel they should each be the size of insert that you predicted from your restriction mapping.

Dilutions
This lab involves doing a serial dilution (see the lambda phage lab) of your isolated plasmid (from lab # 4), setting up 2 PCR reactions with this diluted template, running the PCR in the thermocycler, and then sizing the resultant fragments by agarose gel electrophoresis. This whole procedure should take about 6 hrs., so it will be done over two weeks. The first week, you will do the serial dilutions, set up the reactions and put the reactions in the thermocycler. The next week you will run the reactions out on an agarose gel.
Note: If your insert is greater than 2.0 Kb, tell me and I will give you a different plasmid because this is too large for efficient amplification.

Setting up the Reactions
1. Take three tubes and mark them 10^-2, 10^-4 and 10^-6. Put 199 ul of H₂O in the 10^-2 tube and 990 ul the 10^-4 and 10^-6 tubes. Put 2 ul of your original plasmid in the 10-2 tube and mix well. Put 10 ul of the solution from the 10^-2 tube in the 10^-4 tube and mix well. Put 10 ul of the solution from the 10^-4 tube in the 10^-6 tube and mix well. Each of these is a 100 fold dilution.
2. Label 2 PCR tubes (the 0.65 ml tubes provided) 10^-4 and 10^-6.
3. Set up the following reactions in each of the tubes (I will be adding the buffer mix and Taq polymerase):

       34.5 ul   H2O
        1.0 ul   diluted DNA solution
       14.0 ul   Buffer mix (buffer, primers, MgCl2, and dNTPs)
        0.5 ul   Taq polymerase

       50.0 ul   Total

4. Place these three tubes in the thermocycler.

After you have run the agarose gel, note the extent to which you diluted the DNA in the reactions. Assuming that your original plasmid stock was approximately 1 ug / ul (which is probably a good estimate for most of you), the 10^-6 dilution had a concentration of 1 femtogram / ul. This means that your 10^-6 PCR reaction only has about 1 picogram of DNA template. (Imagine - one picogram!) For this reason, on the agarose gel you should only see the amplified fragment and not the DNA from the original template.

Principle of the PCR

The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.

1. The cycling reactions :
   There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.
   
   1. Denaturation at 94C:
      During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).
   2. Annealing at 54C :
      The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
   3. extension at 72C :
      This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment.
      The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)
   
   
   
   Figure 3 : The different steps in PCR.
2. 
   
   Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on.
   
   
   Figure 4 : The exponential amplification of the gene in PCR.
   
3. Is there a gene copied during PCR and is it the right size ?
   Before the PCR product is used in further applications, it has to be checked if :
   1. There is a product formed.
      Though biochemistry is an exact science, not every PCR is successful. There is for example a possibility that the quality of the DNA is poor, that one of the primers doesn't fit, or that there is too much starting template
   2. The product is of the right size
      It is possible that there is a product, for example a band of 500 bases, but the expected gene should be 1800 bases long. In that case, one of the primers probably fits on a part of the gene closer to the other primer. It is also possible that both primers fit on a totally different gene.
   3. Only one band is formed.
      As in the description above, it is possible that the primers fit on the desired locations, and also on other locations. In that case, you can have different bands in one lane on a gel.
   
   
   Figure 5 : Verification of the PCR product on gel.
   The ladder is a mixture of fragments with known size to compare with the PCR fragments. Notice that the distance between the different fragments of the ladder is logarithmic. Lane 1 : PCR fragment is approximately 1850 bases long. Lane 2 and 4 : the fragments are approximately 800 bases long. Lane 3 : no product is formed, so the PCR failed. Lane 5 : multiple bands are formed because one of the primers fits on different places.