POLYMERASE CHAIN REACTION

1. POLYMERASE CHAIN REACTION

2. DNA Structure • DNA consists of two molecules that are arranged into a ladder-like structure called a Double Helix. • A molecule of DNA is made up of millions of tiny subunits called Nucleotides. • Each nucleotide consists of: • Phosphate group • Pentose sugar • Nitrogenous base

3. Nucleotides Phosphate Nitrogenous Base Pentose Sugar

4. Nucleotides • The phosphate and sugar form the backbone of the ladder, whereas the bases form the “ladder steps”. here are four types of nitrogenous bases.

5. Nucleotides A T C Adenine Thymine G Guanine Cytosine

6. Nucleotides • Each base will only bind with one other specific base. • Adenine (A) • Thymine (T) • Cytosine (C) • Guanine (G) Form a base pair. Form a base pair.

7. The bonds between the bases are hydrogenous bonds. • Three bonds between G & C and two bonds between T & A. • Because of this complementary base pairing, the order of the bases in one strand determines the order of the bases in the other strand.

8. A T A A A T T T C C C G G G

9. To recognize the genetic code found in DNA we need to look at the sequence of bases. • The bases are arranged in triplets called codons. A G G - C T C - A A G - T C C - T A G T C C- G A G - T T C - A G G- A T C

10. A gene is a section of DNA that codes for a protein. • Each unique gene has a unique sequence of bases. • This unique sequence of bases will code for the production of a unique protein. • It is these proteins and combination of proteins that give us a unique phenotype.

11. DNA Gene Protein Trait

12. The polymerase chain reaction (PCR) is a molecular biology technique for isolating and amplifying a fragment of DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast).

13. Polymerase Chain Reaction Polymerase: DNA polymerase • DNA polymerase duplicates DNA. • Before a cell divides, its DNA must be duplicated. Chain Reaction: • The product of a reaction is used to amplify the same reaction. • Results in rapid increase in the product.

14. Applications PCR is now a common technique used in medical and biological research labs for a variety of tasks, such as; • The sequencing of genes and the diagnosis of hereditary diseases. • The identification of genetic fingerprints (used in forensics and paternity testing) • The detection and diagnosis of infectious diseases

15. Basic components • DNA polymerase ( Taq polymerase) • Cannot assemble a new strand from components. Needs a pre-existing DNA to duplicate called “template DNA”. • Can only extend an existing piece of DNA called “primers” • DNA strands are anti-parallel • One strand goes in 5’  3’ • The complementary strand is opposite • DNA polymerase always moves in one direction (from 5’  3’)

16. DNA template that contains the region of the DNA fragment to be amplified. • One or more primers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified.

17. 3’ 5’ dTTP 3’ 5’ dATP dCTP dCTP dATP dTTP dTTP dGTP dCTP dATP dATP dATP dCTP dTTP dGTP dGTP dGTP dATP dTTP dATP dCTP dTTP dGTP dGTP dCTP • Deoxynucleotidetriphosphates, (dNTPs) from which the DNA polymerase builds the new DNA.

18. Buffer solution, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase. • Divalent cation, magnesium or manganese ions. • Monovalentcationpotassium ions.

19. Thermal cycler • The DNA, DNA polymerase, buffer, nucleoside triphosphates, and primers are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler • This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction.

20. PCR usually consists of a series of 30 to 40 cycles. There are three main steps: • Denaturationat 94oC to 98oC for 1 minute. • Annealingat 54°C to 64oC for 45 seconds. • Extension/Elongation at 70°C to 74oC for 2 minute.

21. Main idea • Heating separates the double stranded DNA "Denaturation” • Slow cooling anneals the two strands “Annealing”

22. Denaturation In this step double stranded DNA molecules are melted to yield single stranded DNA (template strand).

23. 2. Annealing • In this step the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template. • Stable bonds are only formed when the primer sequence very closely matches the template sequence. • To this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis.

24. Extension/Elongation • In this step DNA polymerase synthesizes new DNA strands complementary to the DNA template strands. • Primers that have annealed to DNA regions with mismatching bases dissociate from the template and are not extended.

25. PCR amplification

26. Reverse Transcription PCR (RT-PCR) • Is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. • The PCR reaction is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.

27. DETECTION OF AMPLIFICATION PRODUCTS BY GEL ELECTROPHORESIS • Agarose gel electrophoresis is commonly employed for size separation of the PCR products. • This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field. • Shorter molecules move faster and migrate further than longer ones. • The size's of PCR products is determined by comparison with a DNA ladder, which contains DNA fragments of known size, ran on the gel along with the PCR products.

28. Lane 1 : PCR fragment is approximately 1850 bases long. Lane 2 and 4 : the fragments are approximately 800 bases long. Lane 3 : no product is formed Lane 5 : multiple bands are formed because one of the primers fits on different places.

29. Gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several megabases (millions of bases). However, it is normally used in a range of 100 bp to 20 kbp. • Typical run times are about an hour. • Agarose is purified from agar. Different purities of agarose are commercially available with different melting properties. • High purity low melt agarose is often used if the DNA is to be extracted from the gel.

30. After electrophoresis the gel is illuminated with an ultraviolet lamp. • The ethidium bromide fluoresces pink in the presence of DNA. • The DNA band can also be cut out of the gel and can then be dissolved to recover the purified DNA.