Polymerase Chain Reaction Practical
- 1. Practical # 9 19-12-2018 POLYMERASE CHAIN REACTION(PCR) PCRis a laboratory method used for making a very large number of copies of short sections of DNA from a very small sample of genetic material. This process is called "amplifying" the DNA and it enables specific genes of interest to be detected or measured. DNA is made up of repeating sequences of four bases – adenine, thymine, guanine, and cytosine. These sequences form two strands that are bound together in a double helix structure by hydrogen bonds (like a spiral staircase). Each half of the helix is a complement of the other. In humans, it is the difference in the sequence of these bases on each strand of DNA that leads to the uniqueness of each person's genetic makeup. The arrangement of the bases in each gene is used to produce RNA, which in turn produces a protein. There are about 25,000 genes in a human genome, and expression of these genes leads to the production of a large number of proteins that make up our bodies. The DNA of other organisms such as bacteria and viruses is also composed of thousands of different genes that code for their proteins. How is the method performed? PCR is carried out in several steps or "cycles" in an instrument called a thermocycler. This instrument increases and decreases the temperature of the specimen at defined intervals during the procedure. 1. The first step or cycle of PCR is to separate the strands of DNA into two single strands by increasing the temperature of the sample that contains the DNA of interest. This is called "Denaturing" the DNA. 2. Once the strands separate, the sample is cooled slightly and forward and reverse primers are added and allowed to bind to the single DNA strands. Primers are short sequences of bases made specifically to recognize and bind to the section of DNA to be amplified, which are the very specific sequence of bases that are part of the gene or genes of interest this is called Annealing. Primers are called "forward" and "reverse" in reference to the direction that the bases within the section of DNA are copied. 3. After the two primers attach to each strand of the DNA, a DNA enzyme (frequently Taq polymerase) then copies the DNA sequence on each half of the helix from the forward to the reverse primer, forming two double stranded sections of DNA, each with one original half and one new half known as EXTENSION. Taq polymerase is an enzyme found in a bacterium (Thermues aquaticus) that grows in very hot water, such as in geysers or hot springs. Polymerases copy DNA (or RNA) to make new strands. The Taq polymerase is especially helpful for laboratory testing because (unlike many other enzymes) it does not break down at very high temperatures needed to do PCR.
- 2. 4. When heat is applied again, each of the two double strands separate to make four single strands and, when cooled, the primers and polymerase act to make four double strand sections. The four strands becomes eight in the next cycle, eight become sixteen, and so on. 5. Within 30 to 40 cycles, as many as a billion copies of the original DNA section can be produced and are then available to be used in numerous molecular diagnostic tests. This process has been automated so that a billion copies of the original DNA can be produced within a few hours. General recepie and requirements 1. DNA template 50ng/ul 2. Primers Forward and reverse 3. Taq Polymerase 4. dNTPs 5. Monovalent cations ( KCL) 6. Divalent Cations (MgCl2) 7. Buffer Precautions 1. Aovid contamination 2. Tm of primers should be calculated before starting PCR 3. Conc. of template DNA should be kept in mind
- 3. PCR Cycle