2. It is a molecular technology aim to amplify a single or few
copiesoftheDNAtothousandsormillionsofcopies.
Developed in 1983 by Kary Mullis, PCR is now a common
and often indispensable technique used in medical and
biological research labs for a variety of applications. These
include diagnosis of infectious diseases, DNA sequencing
andDNA-basedphylogeny.
In 1993, Mullis was awarded the Nobel prize in Chemistry
alongwith MichaelSmithforhiswork onPCR.
3. • Steps in PCR reaction
1. Denaturation
2. Annealing
3. Extension
• Constituents of PCR
reaction :
1. Target DNA
2. Pair of primers
3. dNTPs
4. Thermostable DNA
Polymerase
5. Mg++ ions
6. Buffer solution
5. It is a special type of the PCR used for
detection of multiple pathogens by using
Multiple primers sets each one targets a
particular pathogen.
Uses:
This permits the simultaneous analysis of
multiple targets in asinglesample.
6.
7. Used to increase the specificity of DNA
amplification.
Two sets of primers are used in two
successive reactions.
In the first PCR, one pair of primers is used to
generate DNA products, which will be the
target forthe second reaction.
8. Using one ('hemi-nesting') or two different
primers whose binding sites are located
(nested) within the first set, thus increasing
specificity.
Uses:Detection of pathogens that occur with very
few amount.
13. Used to measure the specific amount of
target DNA(or RNA) in asample.
By measuring amplification only within the
phase of true exponential increase, the
amount of measured product more accurately
reflects the initial amount of target.
Special thermal cyclers are used that monitor
the amount of product during the
amplification.
14. It is a technique performed manually by
heating the reaction components to the
DNA melting temperature (e.g. 95°C)
before adding thepolymerase.
15. In this type the annealing temperature is
gradually decreased in latercycles.
The annealing temperature in the early cycles is
usually 3-5°C above the standard Tmof the primers
used, while in the later cycles it is a similar
amount below theTm.
The initial higher annealing temperature leads to
greater specificity for primer binding, while the
lower temperatures permit more efficient
amplification at the end of the reaction.
16. It also known as Polymerase Cycling Assembly or PCA
It is a method for the assembly of large
DNA oligonucleotides from shorter
fragments.
It uses the same technology as PCR, but takes
advantage of DNA hybridization and annealing as well
as DNA polymerase to amplify a complete sequence of
DNA in a precise order based on the single stranded
oligonucleotides allows for the production of synthetic
genes and even entire synthetic genomes
17. 1. Inverse PCR
2. Multiplex PCR
3. Hot start PCR
4. Nested PCR
5. In situ PCR
6. Long PCR
7. Colony PCR
8. Real time PCR
9. Touch down PCR
10. Band stab PCR
11. Reverse transcriptase
PCR
12.Degenerate PCR
13. Anchored PCR
14. Asymmetric PCR
15. Assembly PCR
16. Quantitative PCR
17. Methylation specific
PCR
18.Ligation mediated
PCR
19. Allele specific PCR
20. Digital PCR
21.Overlap Extension PCR
22.Solid phase PCR
23.Miniprimer PCR
24. Universal fast
walking PCR
25.VNTR PCR
26.ISSR PCR
27.Semi quantitative
PCR
28.Differential
display reverse
transcriptase PCR
