PCR Group biochemistry practical mbbs second year

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1. PCR and Real time PCR Group No 1

2. Group Members Mobeen Asghar Moin Aslam Jarrar Hussain Azam zamin Fahad Ali

3. PCR Stands for Polymerase chain reaction PCR targets and amplifies a specific region of a DNA strand. It is an in vitro technique to generate large quantities of a specified DNA. Often, only a small amount of DNA is available e.g.. A drop of blood, Semen strains, Single hair, vaginal swabs etc.

4. PCR PCR is fast, sensitive and capable of copying a single DNA sequence of a viable or nonviable cell over a billion times within 3-5 hours . Developed in 1983 by Kary Mullis, PCR is now a common technique used in clinical and research laboratories for a broad variety of applications.

5. Amplification Methods Two methods currently exist for amplifying the DNA or making copies Cloning—Very time consuming process PCR—Works on even a single molecule quickly

6. Requirements for PCR DNA template: DNA template is DNA target sequence. DNA template is the DNA molecule that contains the DNA region (segment) to be amplified, the segment we are concerned which is the target sequence. DNA polymerase: DNA polymerase sequentially adds nucleotides complimentary to template strand at 3’-OH of the bound primers and synthesizes new strands of DNA complementary to the target sequence. The most commonly used DNA polymerase is Taq DNA polymerase (from Thermus aquaticus , a thermophilic bacterium) because of high temperature stability.

7. Requirements of PCR Primers: Primers are synthetic DNA strands of about 18 to 25 nucleotides complementary to 3’ end of the template strand. DNA polymerase starts synthesizing new DNA from the 3’ end of the primer . Nucleotides ( dNTPs or deoxynucleotide triphosphates) All types of nucleotides are "building blocks" for new DNA strands and essential for reaction. It includes Adenine(A), Guanine(G), Cytosine(C), Thymine(T) or Uracil(U).

8. Requirements of PCR Magnesium As a co-factor Buffer solution For maintenance of pH

9. Principle of PCR The DNA intended to be amplified is denatured. Each strand is then subjected to reanneal with primers (renaturation ). Primers are segments of 20-30 nucleotide length which are complementary to flanking regions of target DNA. Primer template combine is then used for DNA synthesis involving the enzyme, DNA polymerase. Amplification is achieved with repeated cycles .

10. Steps of PCR Step 1 – Denaturation Step 2 – Renaturation ( annealing) Step 3 – Polymerization (synthesis) These steps in a PCR 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.

11. Denaturation The reaction mixture is heated to a temperature between 90-98° C so that the ds DNA is denatured into single strands by disrupting the hydrogen bonds between complementary bases. Duration of this step is 1-2 mins

12. Annealing Temperature of reaction mixture is cooled to 45-60°C (54 ° C ) Primers base pair with the complementary sequence in the DNA. Hydrogen bonds reform .

13. Extension The temperature is now shifted to 72° C which is ideal for polymerase. Primers are extended by joining the bases complementary to DNA strands E longation step continues where the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template. Now first cycle is over and next cycle is continued ,as PCR machine is automated thermocycler the same cycle is repeated upto 30-40 times

14. Applications of PCR Diagnosis Cancer detection Prenatal diagnosis Genetic disorders PCR in DNA sequencing Fossil studies

15. Applications of PCR Used in forensic science and DNA fingerprinting Organ transplant Sex determination PCR in genotology

16. Real Time PCR (qPCR or qrtPCR ) Real time PCR is a type of PCR which is used to amplify and simultaneously quantify a target DNA molecule. In Real time PCR amount of amplification is known at any given point of time. In the usual PCR the amplification is known at the end of the PCR qPCR uses flouroscent dyes such as SYBR green or flurophore containing DNA probes such as Taq Man, to measure the amount of amplified product in real time.

17. Therapeutics study PCR can be used to help diagnose Viruses. It's also useful after treatment to see if copies of the mutation gene are still there. If copies of this gene are found it means that the disease is still present, even when the cells can't be seen with a microscope.

18. Thank You