polymerase chain reaction (PCR) technique

dr esraa allam clinical pathology department

1. Polymerase chain PCR) ) reaction by Esraa Tawfik Allam

2. DNA

3. DNA stands for deoxyribose nucleic acid This chemical substance is present in the nucleus of all cells in all living organisms DNA controls all the chemical changes which take place in cells DNA

4. DNA is a very large molecule made up of a long chain of sub-units The sub-units are called nucleotides Each nucleotide is made up of a sugar called deoxyribose a phosphate group -PO 4 and an organic base DNA molecule

5. The most common organic bases are Adenine (A) Thymine (T) Cytosine (C) Guanine (G) The bases

6. DNA is a double stranded molecule consists of 2 polynucleotide chains running in opposite directions, antiparallel to each other. The bases are on the inside of the molecules and the 2 chains are joined together by double H-bond between A and T and triple H-bond between C and G. The base pairing is very specific which make the 2 strands complementary to each other . .

7. PCR, polymerase chain reaction , is a technique used in  molecular biology  to  amplify a single copy or a few copies of a piece of  DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR

8. 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 Before PCR, DNA extraction and isolation from the cell must be occur first

9. Many different methods are available for the isolation of genomic DNA: - Colum extraction - Salt Precipitation - Phenol Chloroform Extraction - Ethanol Precipitation

10. All methods involve : A. disruption and lyses of the starting material followed by B. Removal of proteins and other contaminants and finally C. Recovery of the DNA

11. 1- Cell lysis , to expose the DNA within . 2- removing membrane lipids by adding a detergents or surfactants. 3- removing proteins by adding a protease . 4- removing RNA by adding an Rnase . 5- precipitating the DNA with alcohol- usually ice cold ethanol. In these alcohols , DNA strand will aggregate together, giving a pellet upon centrifugation . This step also removes alcohol- soluble salt. 6-Wash the resulting DNA pellet with alcohol 7-Solubilize the DNA in a slightly alkaline buffer Summary of DNA extraction :

12. 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 5-MgCl 2 cofactor for DNA polymerase 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme PCR-Reaction Components

13. -2 sets of primers (forward & Reverse) Generally 20-30 nucleotides long -Synthetically produced -complimentary to the 3’ ends of target DNA not complimentary to each other -40-60 % GC content preferred for better annealing Primer

14. DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand Usually Taq Polymerase or anyone of the natural or Recombinant thermostable polymerases Stable at T up to 95 C High processivity DNA Polymerase

15. Comprised of 3 steps: -Denaturation of DNA at 95 C -Primer hybridization ( annealing) at 50 -60 C - DNA synthesis ( Primer extension) at 72 C The PCR Cycle

16. -Gel electrophoresis -Sequencing of amplified fragment -Southern blot etc... Detection of amplification products

17. It is a method for separation and analysis of macromolecules ( DNA, RNA and proteins ) and their fragments, based on their size and charge . Nucleic acid molecules are separated by applying an  electric field  to move the negatively charged molecules through a matrix of  agarose  or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving Gel Electrophoresis

18. DNA Ladder Can be used to estimate Size –- Migration of the Bands Quantity – Relative Intensities of the bands Isolate particular (required) DNA band

19. RFLP , is a technique that exploits variations in homologous DNA sequences. It refers to a  difference between samples of  homologous DNA molecules from differing locations of restriction enzyme sites. In   RFLP analysis , the DNA sample is broken into pieces and (digested) by restriction enzymes and the resulting  restriction fragments  are separated according to their lengths by gel electrophoresis. Restriction fragment length polymorphism

20. -Genome mapping and gene function determination -Diagnostics ( prenatal testing of genetic diseases, early detection of cancer, viral infections...) -Detection of drug resistance genes -Forensic (DNA fingerprinting) Applications

21. - fast, reliable (reproducible) results -Contained :(less chances of contamination) -High output -Sensitive -Broad uses Advantages

22. Thank you