The polymerase chain reaction (PCR)

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

What is PCR? Definition Is a powerful and sensitive technique to amplify a piece of DNA very rapidly outside a living cell (DNA amplification in vitro).

PCR Applications PCR is now a common and often indispensable (main) technique used in medical and biological research labs for a variety of applications. Structural analysis Mapping Disease detection Sequencing Cloning Forensic medicine Mutation analysis Scientific research Pre-natal diagnosis

How does PCR work? 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.

Denaturation The initial step denatures the target DNA (H-bonds are broken between strands of DNA with heat) by heating it to 94°C or higher. In the denaturation process, the two intertwined (coiled) strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermo stable DNA polymerase.

Annealing: In this step the temperature is reduced to approximately 50–60°C. At this temperature, the oligonucleotide primers attach to complementary sequences of single stranded DNA. Extension: Finally, the synthesis of new DNA, the DNA polymerase attaches to primer with ssDNA and extends DNA fragment, this temperature is in the range of 70–74°C. The next cycle begins with a return to 94°C for denaturation.

PCR

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) 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 Use of high quality, purified DNA templates greatly enhances the success of PCR reactions. Approximately 104 copies of the target DNA “this means a 1–10 µg/ml of genomic templates” are required to detect a product in 25–30 cycles of PCR. DNA can be a single gene part of a gene 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, 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.

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: the short DNA molecules sequence to be amplified. Oligonucleotide primers Generally 20–30 nucleotides in length, and ideally have a GC content of 40–60%, with GC residues spaced evenly within the primer. 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.

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

4) 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 catalyzes the primer-dependent incorporation of nucleotides into duplex DNA in the 5′→3′ direction in the presence of Mg2+ Magnesium: a necessary cofactor for DNA polymerase activity. A magnesium concentration of 1.5–2.0 mM is optimal for most PCR products generated with Taq DNA Polymerase. Reaction buffer: most often a Tris-based buffer, and salt, commonly KCl.

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. 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: Completed Amplification Cycle n cycles will give 2n copie.

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.

Polymerase Chain Reaction Controls 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

Procedure 1- Prepare master Mix 2- Program the thermocycler 3- Run the samples on thermocycler 4- Analysis of PCR products Loading Dye: Samples are prepared with loading dye and then loaded on the gel. It is used to prepare DNA markers and samples for loading on agarose gels. It contains bromophenol blue dye, for visual tracking of DNA migration during electrophoresis. The presence of glycerol in the solution ensures that the sample sinks at the bottom of the well.

Target DNA Amplification of part of the Human growth hormone gene Specific primers used Forward primer: 5’- TCCCTTCCCAACCATTCCCTTA-3’ Reverse primer: 5’-CCACTCACGGATTTCTGTTGTGTTTC-3’ During Polymerase Chain Reaction (PCR) the primers will be extended from the 3’-end. I know they both anneal to 3' ends of different, complementary DNA strands, for example FP binds to 5'-->3' strand and RP bind to the other ??

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. There should be a 400 bp band for the positive samples. -ve +ve Sample Samples are prepared with loading dye Loading Dye: It contains bromophenol blue dye, for visual tracking of DNA migration during electrophoresis. The presence of glycerol in the solution ensures that the sample sinks at the bottom of the well. يجب ان تظهر المادة الوراثية على هيئة قطعة واحدة فقط, اذا ظهرت على هيئة مسحة يدل على حدوث تلوث اثناء عملية استخلاص المادة الوراثية. Primary: يظهر اسفل قطعة المادة الوراثية

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