1. PCR is used in; Cloning into plasmid vectors DNA sequencing Genetic screening DNA based phylogeny Functional analysis of genes Identification of DNA fingerprints Molecular diagnosis of genetic diseases

2. Step.1.Denaturation Heating to 90-95°C denatures DNA into single strands A standart time of 30 to 60 seconds

3. Step.2 Annealing Temperature is btw 50 and 70 °C Primers (I5-30 nucleotides long) anneal to single-stranded DNA Time for cooling is 30 to 60 seconds Annealing temp. -depend on GC content of primers -3-5 °C below Tm of primers Low-annealing temp-----unspecific primer annealing High annealing temp----prevent annealing and DNA synthesis

4. Step.3. Extension DNA synthesis at temp. btw 70-75 °C Tag DNA polymerase extends primers in 5‘ to 3‘ by adding dNTPs Each set of 3 steps is a ‘cycle’ Each cycle takes about 5 min.(depend on DNA polymerase used,and the length of DNA fragment) 25-30 cycle result in increase in the amount of DNA

5. Initial Denaturation For few minutes for complete denaturation For complex templates or templates with high GC content Final Extension For 5 to I0 min. at 70-75 °C To ensure any remaining single-stranded DNA is fully extended Final Hold At 4-I5 °C

6. Elements for standart PCR reaction DNA polymerase DNA template Primers dNTP MgCl Buffer

7. DNA Polymerase Most widely available form Thermus aquaticus ( Tag ) Optimum polymerization temp. 70-80 °C ≥90 °C loses its activity but not denatured Used 1 to 5 units in reaction (depend on template DNA or primer) High processivity (activity of up to I50 bases per second per molecule At low temp. activity reduced 2 bases per second per molecule Low replication fidelity Lacks 3‘ to 5‘ exonuclease proof-reading activity

8. Primers Forward and reverse primers are needed Optimal primers 20-30 bases in length GC content similar to target DNA should not contain bases complementary to each other should not contain secondary structures,simple repeats Usually 0.1 to 1 µM Too much primer----false initiations,primer-dimer formation

9. dNTP ≤ 200 mM A,T,C,G in equivalent amounts Excess of nucleotide----inhibit enzyme activity, false products Magnesium clorur Cofactor for DNA polymerase It may affect; -primer annealing, product specifity -formation of primer-dimer artifact,enzyme fidelity -temp. for strand dissociation of template Few----low PCR yield High---non-specific product For Tag; I-4 mM at 70-80 °C Template DNA Absolutely pure

10. Buffer standart I0 to 50 mM Tris-Hcl for proper function of enzyme Components to stabilize enzyme: Gelatine Bovine serum albumin Tween20, TritonXI00 (non-ionic detergents) Template DNA with high GC content---- acetamide,glycerol, DMSO

11. RAPD PCR is used to amplify a known sequence Target sequence is determined, primers are designed

12. In RAPD; Target sequence is unknown Primer with arbitrary sequence (I0 base-pair) Primers bind somewhere in the sequence Random segments of large template DNA is amplified

13. Finding Differences Between Genomes Using RAPD Analysis For another DNA template suppose there is a change in annealing site 2 Product A is not produced,product B is produced

14. If you run the 2 RAPD PCR reaction products on agorose gel

15. Reaction conditions: DNA5 µL Primer1 µL dNTP1.2 µL Mgcl21.2 µL Buffer1.5 µL Tag polymerase0.2 µL H2O4.9 µL

16. PCR cycling schedule stepTemperatureTimeCycleDescription 194 °C30 sec1Initial denaturation 294 °CI0 sec 35 Denaturation 35 °C30 secAnnealing 72 °C30 secExtension 372 °C1 min1Final extension