MLAB 2479 Molecular Diagnostics Techniques

MLAB 2337 Molecular Diagnostics Techniques Laboratory 6: Polymerase Chain Reaction

Objectives 1. Briefly describe the purpose of the polymerase chain reaction (PCR). 2. State the name of the individual who developed the PCR technique. 3. State what a “primer” is. 4. State the name of the heat resistant DNA polymerase used in PCR and how it works in the reaction. 5. List the 7 components of the PCR master mix and the purpose of each. 6. List and describe the 6 steps in the PCR and the purpose of each. 7. List the 3 steps of the PCR that are the heart of the reaction. 8. List and describe 6 potential errors which may inhibit or cause failure of a PCR. 9. List 3 items which may be altered when no amplification occurs in a PCR.

Introduction:

The polymerase chain reaction (PCR) is a scientific technique for amplifying DNA sequences in vitro by separating the DNA into two strands and incubating it with oligonucleotide primers and DNA polymerase. It can amplify (copy) a specific sequence of DNA by as many as one billion times and is important in biotechnology, forensics, medicine, and genetic research.

This technique was developed in 1983 by Kary Mullis when he was working for Cetus. That spring, according to Mullis, he was driving his vehicle late one night with his girlfriend, who was also a chemist at Cetus, when he had the idea to use a pair of primers to bracket the desired DNA sequence and to copy it using DNA polymerase, a technique which would allow a small strand of DNA to be copied almost an infinite number of times. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.

Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. A complication at this point is that the DNA polymerase used was destroyed by the high heat used at the start of each replication cycle and had to be replaced. In 1986, Mullis started to use Thermophilus aquaticus (Taq) DNA polymerase to amplify segments of DNA. The Taq polymerase was heat resistant and would only need to be added once, thus making the technique dramatically more affordable and subject to automation. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. This has created revolutions in biochemistry, molecular biology, genetics, medicine and forensics.

Lab 6: Polymerase Chain Reaction Page 1 Taq polymerase is a DNA polymerase which enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Principle

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents called the “master mix”. These components include:

1. DNA template which will be amplified by the PCR reaction. 2. Two primers which are short pieces of DNA (20-30 bases) that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. These bind to the template DNA allowing TAq DNA polymerase enzyme to initiate incorporation of the deoxynucleotides. Both specific and universal primers can be used. 3. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C. This is a a heat stable enzyme that adds the deoxnucleotides to the DNA template. 4. Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups). These are the building-blocks from which the DNA polymerase synthesizes a new DNA strand. It is important that equal amounts of each nucleotide (dATP, dTTP, dCTP and dGTP) are added to the master mix to prevent mismatches of bases. 5. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. 6. Magnesium is a required cofactor for thermostable DNA polymerase. Divalent cations, magnesium or manganese ions may be used. Mg2+ in the PCR mixture stabilizes the dsDNA and raises the Tm and is important for controlling the specificity of the reaction. Generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis. 7. Potassium chloride (KCl) is used to improve the PCR amplification of DNA fragments, especially those from 100-1000bp. For amplification of long products (greater than 1000bp) a lower salt concentration is needed, for short products (less than 1000bp) a higher salt concentration is best.

The master mix buffer can be stored at room temperature. The deoxynucleotides, primers and Taq DNA polymerase enzyme must be stored at -20C

Lab 6: Polymerase Chain Reaction Page 2 The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. The following lists the steps in each PCR cycle:

1. Initialization step : This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. 2. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. 3. Annealing step : The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation . 4. Extension/elongation step : The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. 5. Final elongation : This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. 6. Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

Steps 2-4 (denaturation, annealing and extension) are the heart of the PCR procedure.

Lab 6: Polymerase Chain Reaction Page 3 PCR Sources of Error

There are many errors which can inhibit or cause failure of a PCR.

1. Template DNA - The amount of DNA in a PCR directly affects the outcome. a. Using too much total DNA can lead to false priming and poor DNA synthesis. It may also inhibit PCR because all of the primers will be used up early on. b. Too little target DNA increases chance of loss due to clotting, adsorption or chemical or enzymatic degradation. The concentration of the target DNA should be balanced with the number of cycles. 2. dNTP Concentration a. Excessive concentrations can inhibit the PCR preventing the formation of product. b. Low concentrations may cause incomplete primer elongation or premature termination of DNA synthesis during the elongation step of the PCR cycle. 3. Primer Concentration a. High concentrations of primers capable of forming dimers may result in the creation of primer-dimers. b. High concentrations for primers not capable of primer-dimer may lead to non-specific primer binding and creation of undesirable PCR products. 4. Taq Content a. Inadequate amount may cause incomplete primer elongation or premature termination of the PCR product synthesis during the elongations step of a PCR cycle. b. Too much Taq may result in an increased amount of unwanted DNA fragments (smears on a gel) while a huge increase may cause the reaction to fail with no detectable product produced. 5. Magnesium Concentration a. Too low requires more stringent base pairing in the annealing step. Too few Mg2+ ions may result in a low yield of PCR product or cause reaction to fail. b. Too high a concentration may increase the yield of non-specific products and promote misincorporation. May result in smearing or extraneous bands to form. c. A major change in the concentration of dNTP in a reaction will require a change in the concentration of MgCl2. d. Any changes made to the KCl-based buffer concentration or any other component of the PCR mix may require adjustment in the concentration of Mg2+. 6. Potassium Chloride Concentration a. Low concentration may result in long, unwanted products. b. High concentrations may produce unwanted non-specific short products.

Lab 6: Polymerase Chain Reaction Page 4 No Amplification Check List

If problems are encountered the procedure must be repeated and the following check list should be followed:

1. Make sure polymerase buffer is fully thawed and well mixed. 2. Make sure primers are diluted to correct concentration. 3. Make up a new dNTP solution. dNTPs can be destroyed by repeated freeze-thaw cycles. 4. Make up new DAN template, especially if working with genomic DNA. Old stock may be degraded or sheared. 5. Change annealing temperature. If temperature is too high there will be no priming at the desired sequence. If the annealing temperature is too low it may result in non-specific priming. 6. Try reactions with varying template concentrations. 7. Check the cycler. 8. Use a different cycler.

Lab 6: Polymerase Chain Reaction Page 5 Name ______Date ______

MLAB 2337 Molecular Diagnostics Techniques Laboratory 6: Polymerase Chain Reaction Study Questions Points: /31 Instructions:  Copy/paste the following study questions into word processing document.  Answer the questions  Save the file as “Lab6PCR_YOURNAME”  Submit to “Assignments” in BlackBoard

1. Briefly describe the purpose of the polymerase chain reaction (PCR). (2 points)

2. State the name of the individual who developed the PCR technique. (1 point)

3. State what a “primer” is. (1 point)

4. State the name of the heat resistant DNA polymerase used in PCR and how it works in the reaction. (2 points) a. b.

5. List the 7 components of the PCR master mix and the purpose of each. (7 points) a. b. c. d. e. f. g. 6. List and describe the 6 steps in the PCR, including temperature if required, and the purpose of each. (6 points) a. b. c. d. e. f. 7. List the 3 steps of the PCR that are the heart of the reaction. (3 points) a. b. c.

Lab 6: Polymerase Chain Reaction Page 6 8. List 6 potential sources of error which may inhibit or cause failure of a PCR and give an example of each. (6 points) a. b. c. d. e. f. 9. List 3 items which may be altered when no amplification occurs in a PCR. (3 points) a. b. c.

Lab 6: Polymerase Chain Reaction Page 7