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

• 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 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

• 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

• 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. 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

•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

• 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

• 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:

DNA 5 µL

Primer 1 µL

dNTP 1.2 µL

Mgcl2 1.2 µL

Buffer 1.5 µL

Tag polymerase 0.2 µL

H2O 4.9 µL

•PCR cycling schedule

step Temperature Time Cycle Description

1 94 °C 30 sec 1 Initial denaturation

2 94 °C I0 sec

35

Denaturation

35 °C 30 sec Annealing

72 °C 30 sec Extension

3 72 °C 1 min 1 Final extension