The polymerase chain reaction (PCR)

Experiment Goals Understand how PCR technique works Perform PCR experiment Analyze PCR products

What is PCR? Definition The polymerase chain reaction (PCR) is a technique to amplify a piece of DNA very rapidly outside a living cell

Development of PCR 1971: Khorana described basic principle of DNA replication using DNA primers 1983: Dr. Karry Mullis developed PCR technique, for which he received the Nobel Prize in Chemistry in 1993.

PCR Applications PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. Structural analysis Mapping DNA typing Site-directed mutagenesis Disease detection Sequencing Cloning Forensic medicine Mutation analysis Scientific research Detection of gene expression Pre-natal diagnosis

How does PCR work? DNA is denatured (H-bonds are broken between strands of DNA with heat), 94oC Primers attach to complementary sequences of single stranded DNA, 55-60oC DNA polymerase attaches to primer with ssDNA and extends DNA fragment, 72oC Thus, making double stranded DNA This is done by changing the temperature

PCR Reaction Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction Oligonucleotides are short sequences of nucleotides (RNA or DNA), typically with twenty or fewer bases. Automated synthesizers allow the synthesis of oligonucleotides up to 160 to 200 bases. Oligonucleotides composed of DNA (deoxyoligonucleotides) are often used in the polymerase chain reaction (PCR), a procedure that can be employed to amplify almost any piece of DNA 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

1) Target DNA Target of DNA can be a single gene part of a gene or a non-coding sequence

DNA Quality DNA should be intact and free of contaminants that inhibit amplification. Contaminants can be purified from the original DNA source. Heme from blood, and melanin from hair Contaminants can be introduced during the purification process. Phenol, ethanol, sodium dodecyl sulfate (SDS) and other detergents, and salts. Contaminants may be purified from the original source (e.g., the tissue from which DNA was isolated). For example, heme from blood, humic acid from soil and melanin from hair can copurify with DNA and inhibit amplification. Also, contaminants can be introduced during the purification process. An easy way to detect inhibitors is to add (spike) the DNA template in question into a positive control reaction, a reaction which is known to amplify well. If the spiked control reaction fails, the template contains an inhibitor and needs additional purification before amplification. Alternatively, a smaller volume of DNA can be added to the PCR in hopes that the inhibitor will be diluted to a level where it no longer interferes with amplification.

DNA quantity More template is not necessarily better. Too much template can cause nonspecific amplification. Too little template will result in little or no PCR product.

How Big A Target is? Amplification products are typically in the size range 100-1500 bp. Longer targets are amplifiable >25 kb. Requires modified reaction buffer, cocktails of polymerases, and longer extension times.

2) Pair of Primers Primers define the DNA sequence to be amplified—they give the PCR specificity. Primers bind (anneal) to the DNA template and act as starting points for the DNA polymerase, DNA polymerases cannot initiate DNA synthesis without a primer. The distance between the two primers determines the length of the newly synthesized DNA molecules.

3) dNTPs (deoxynucleotidetriphosphates) The building blocks for the newly synthesized DNA strands. dATP, dGTP, dCTP or dTTP

4) Thermostable DNA Polymerase DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring This enzyme is heat-tolerant it is thermally tolerant (survives the melting T of DNA denaturation) which also means the process is more specific, higher temps result in less mismatch – more specific replication

Running PCR The PCR is commonly carried out in a reaction volume of 15-100 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler allows heating and cooling of the reaction tubes to control the temperature required at each reaction step. 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.

Initialization step Prior to the first cycle, there is an initialization step the PCR reaction is often heated to a temperature of 94-96°C, and this temperature is then held for 1-9 minutes This first hold is employed to ensure that most of the DNA template and primers are denatured, Also, some PCR polymerases require this step for activation

PCR Reaction Cycles One PCR cycle consists of a DNA denaturation step, a primer annealing step and a primer extension step. DNA Denaturation: Expose the DNA template to high temperatures to separate the two DNA strands and allow access by DNA polymerase and PCR primers. Primer Annealing: Lower the temperature to allow primers to anneal to their complementary sequence. Primer Extension: Adjust the temperature for optimal thermostable DNA polymerase activity to extend primers. Annealing Temperature, Tanneal – the temperature at which primers anneal to the template DNA. It can be calculated from Tm . Tanneal = Tm_primer – 4C

PCR

PCR: First 4 Cycles

PCR: Completed Amplification Cycle

PCR: Completed Amplification Cycle Each cycle: 1 copy of DNA template will give 2 copies from double-stranded DNA templates. n cycles will give 2n copies Assuming a cycle lasts 6 min: 1 double-stranded DNA molecule 35 cycles 34x109 copies in 3.5 hrs! There are also ≈ 60 other DNA copies

Analyze PCR products Check a sample by gel electrophoresis. Is the product the size that you expected? Is there more than one band? Is any band the correct size? May need to optimize the reaction conditions.

PCR Modifications Nested PCR Multiplex PCR Reverse-transcriptase PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. Multiplex PCR The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. Reverse-transcriptase PCR (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.

Polymerase Chain Reaction Controls for PCR Blank reaction (Negative control reaction) Controls for contamination Contains all reagents except DNA template Positive control reaction Controls for sensitivity Contains all reagents and a known target-containing DNA template

Contamination of PCR Reactions Most common cause is carelessness and bad technique. Separate pre- and post-PCR facilities. Dedicated pipettes and reagents. Change gloves. Aerosol barrier pipette tips. 10% bleach, UV light

Procedure 1- Prepare master Mix 2- Program the thermocycler 3- Run the samples on thermocycler 4- Analysis of PCR products

Target DNA Amplification of part of the Human growth hormone gene Specific primers used Forward primer: 5’- TCCCTTCCCAACCATTCCCTTA-3’ Reverse primer: 5’-CCACTCACGGATTTCTGTTGTGTTTC-3’

1- Master Mix PCR reaction mixture Reagent Volume (µl) Final concentration PCR buffer (X10) 2.0 10 mM MgCl2 (25 mM) 1.6 2.0 mM dNTPs (100mM) 0.1 0.1 mM Primer 1 (F) 0.2 1.0 µM Primer 2 (R) Taq DNA polymerase 0.25 2.0 U DNA template 100 ng Water 13.7 102 - 105 copies of template

2- Program the Thermocycler The Thermocycler Profile is: Step 1: Denaturation for 3 min. at 95oC Step 2: 35 cycles Melting for 60 sec. at 95oC Annealing for 60 sec. at 57oC Extension for 90 sec. at 72oC Step 3: Final elongation for 10 min. at 72oC

4- Analysis of PCR products Analyse products on 2% agarose gel containing ethidium bromide Visualize the PCR product on UV transilluminator -ve +ve Sample