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

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

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

Step.3. Extension DNA synthesis at temp. btw °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) cycle result in increase in the amount of DNA

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 °C To ensure any remaining single-stranded DNA is fully extended Final Hold At 4-I5 °C

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

DNA Polymerase Most widely available form Thermus aquaticus ( Tag ) Optimum polymerization temp °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

Primers Forward and reverse primers are needed Optimal primers 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

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 °C Template DNA Absolutely pure

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

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

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

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

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

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

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