pcr is used in; cloning into plasmid vectors dna sequencing genetic screening dna based phylogeny...
TRANSCRIPT
• 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
•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