pcr, polymerase chain reaction, Asymmetric PCR Nested PCR Multiplex PCR Competitive PCR RT-PCR (reverse transcriptase) Real-time PCR Hot start PCR Inverse PCR

1. Polymerase Chain Reaction
(PCR) and Its Applications
Dr. Dinesh Jain
Associate Professor
SMS Medical College Jaipur

3. What is PCR?
It was invented in 1983 by Dr. Kary
Mullis, for which he received the Nobel
Prize in Chemistry in 1993.
PCR is an exponentially progressing
synthesis of the defined target DNA
sequences in vitro.

4. What is PCR? :
Why “Chain”?
It is called “chain” because the
products of the first reaction become
substrates of the following one, and
so on.

5. What is PCR? :
The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that
catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength
of the reaction solution suitable for the activity of
the enzyme

6. 1. Hot-Start PCR
2. Inverse PCR
3. Multiplex PCR
4. Nested PCR
5. Ligation-Mediated PCR
6. Methylation-Specific PCR (MSP)
7. Multiplex Ligation-Dependent
Probe Amplification (MLPA)
8. Thermal Asymmetric Interlaced
PCR (Tail- PCR)
9. Assembly PCR
10. Asymmetric PCR
11. Colony PCR
12. Helicase-dependent amplification
13. In Situ PCR (ISH)
14. Intersequence-specific PCR (ISSR)
15. Ligation-mediated PCR
16. Methylation-specific PCR (MSP)
17. Long PCR
18. Miniprimer PCR
19. Overlap-extension PCR
20. Quantitative PCR (Q-PCR)
21. Reverse Transcription PCR (RT-
PCR
22. Solid Phase PCR
23. Touchdown PCR (Step-down PCR)
24. Universal Fast Walking
25. Variable Number of Tandem
Repeats (VNTR) PCR
26. InterSequence-Specific PCR (or
ISSR-PCR)
Types of PCR

7. The Reaction
THERMOCYCLER
PCR tube

9. DNA copies vs Cycle number
0
500000
1000000
1500000
2000000
2500000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Cycle number
DNA
copies

10. Applications of PCR
• Classification
of organisms
• Genotyping
• Molecular
archaeology
• Mutagenesis
• Mutation
detection
• Sequencing
• Cancer research
• Detection of
pathogens
• DNA
fingerprinting
• Drug discovery
• Genetic
matching
• Genetic
engineering
• Pre-natal
diagnosis

11. Applications of PCR
Basic Research Applied Research
• Genetic matching
• Detection of pathogens
• Pre-natal diagnosis
• DNA fingerprinting
• Gene therapy
• Mutation screening
• Drug discovery
• Classification of organisms
• Genotyping
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies

12. Applications of PCR
Molecular Identification Sequencing Genetic Engineering
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
• Bioinformatics
• Genomic cloning
• Human Genome Project
• Site-directed mutagenesis
• Gene expression studies

13. Steps in PCR
Initialization
Denaturation
Annealing
Extension / Elongation
Final elongation
Final hold
Initialization step
Heating the reaction to a temperature of
 94-96°C for 1-9 minutes.

14.  Denaturation step
 94-98°C for 20-30 seconds.
 Denaturation of DNA template by disrupting the hydrogen
bonds between complementary bases of the DNA strands,
yielding single strands of DNA.

17.  Annealing step
 50-65°C for 20-40 seconds
 Stable DNA-DNA hydrogen bonds are formed
 The polymerase binds to the primer-template hybrid and
begins DNA synthesis.

19. Extension/elongation step
 75-80°C
 At this step the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template by adding
dNTPs in 5' to 3' direction.

20. Final elongation
 70-74°C for 5-15 minutes
 To ensure that any remaining single-stranded DNA is
fully extended.
Final hold
 4-15°C for an indefinite time
 short-term storage of the reaction

23. Allele- Specific PCR
• Selective PCR amplification of the alleles to detect single
nucleotide polymorphism (SNP)
• Selective amplification is usually achieved by designing a
primer such that the primer will match or mismatch one of
the alleles at the 3’ end of the primer.

24. Asymmetric PCR
• It is used for DNA sequencing
• The two primers are used in the 100:1 ratio so that after
20-25 cycles of amplification one primer is exhausted thus
single stranded DNA is produced in the next 5-10 cycles

25. Real Time PCR
• Quantitative real time PCR (Q-RT PCR)
• It is used to amplify and simultaneously quantify a target
target DNA molecule
 Real time PCR using DNA dyes
 Fluorescent reporter probe method

26. Real Time PCR

27. Helicase-dependent amplification
 Constant temperature is used rather than cycling through
denaturation and annealing/extension cycles.
 DNA Helicase, an enzyme that unwinds DNA, is used in
place of thermal denaturation.

28. Intersequence-specific PCR (ISSR):
A PCR method for DNA fingerprinting that amplifies
regions between some simple sequence repeats to
produce a unique fingerprint of amplified fragment
lengths.

29. Inverse PCR
 A method used to allow PCR when only one internal
sequence is known.
 This is especially useful in identifying flanking sequences
of various genomic inserts.

31. Anchored PCR
• When sequence of only one end of the desired segment of
gene is known,the primer complimentary to the 3' strand of
this end is used to produce several copies of only one
strand of the gene.

33. RT-PCR (Reverse Transcription PCR)
 It is used to amplify, isolate or identify a known sequence
from a cellular or tissue RNA.
 RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify the
sequence of an RNA transcript.
RACE-PCR
 Used to obtain 3' and 5' end sequence of cDNA transcripts

35. Parameter PCR Gene cloning
1. Final result Selective amplification of
specific sequence
Selective amplification of
specific sequence
2. Manipulation In vitro In vitro and in vivo
3. Selectivity of the specific
segment from complex DNA
First step Last step
4. Quantity of starting material Nanogram (ng) Microgram (m)
5. Biological reagents required DNA polymerase
(Taq polymerase)
Restriction enzymes,
Ligase, vector. bacteria
6. Automation Yes No
7. Labour intensive No Yes
8. Error probability Less More
9. Applications More Less
10. Cost Less More
11. User’s skill Not required Required
12. Time for a typical experiment Four hours Two to four days
Comparison PCR - Polymerase Chain Reaction and Gene Cloning

36. Application of PCR
Cloning a Gene encoding a known protein
Amplification of old DNA
Amplifying cloned DNA from Vectors
Rapid Amplification of cDNA ends
Detecting Bacterial or Viral Infection
● AIDS infection
●Tuberculosis (Mycobacterium tuberculosis)

37. Genetics Diagnosis
Diagnosing inherited disorders
 Cystic fibrosis
 Muscular dystrophy
 Haemophilia A and B
 Sickle cell anaemia
Diagnosing cancer
Blood group typing.

38. Problems with PCR
• Polymerase errors
Polymerase lacks exonuclease activity
• Size limitations
PCR works readily with DNA of lengths two to three
thousand basepairs
• Non specific priming

39. RT-PCR
The enzyme reverse transcriptase is used to make a DNA copy (cDNA) of an
RNA template from a virus or from mRNA.
Viral RNA Bacterial mRNA
AAAA
3’
Protozoan (eukaryotic) poly A mRNA
Primer
Reverse transcriptase
RNA
3’
5’
5’
Extension
c
D
N
A
R
N
A
3
’
3
’
5
’
5
’
Normal PCR with two primers

40. Multiplex PCR
Use of multiple sets of primers to detect more than one organism or to detect
multiple genes in one organism. Remember, the PCR reaction is inherently biased
depending on the G+C content of the target and primer DNA. So performing
multiplex PCR can be tricky.
E. Coli
genome
Salmonella sp.
genome
or

41. Seminested PCR
Three primers are required, the normal upstream and downstream primers as well
as a third, internal primer. Two rounds of PCR are performed, a normal PCR with the
upstream and downstream primer, and then a second round of PCR with the
downstream and internal primer. A second smaller product is the result of the
second round of PCR.
Internal primer
Downstream primer
Upstream primer

42. ICC-PCR
Integrated cell culture PCR is used for virus detection. Cell culture takes 10 – 15 days.
PCR alone detects both infectious and noninfectious particles. So use a combination of
these techniques: grow the sample in cell culture 2 – 3 days, release virus from cells
and perform PCR. This results in the detection of infectious virus in a shorter time with
a 50% cost savings. It also allows use of dilute samples which reduces PCR inhibitory
substances.

43. SYBR Green I
hn
ssDNA -- unbound dye
minimal fluorescence
hn
dsDNA -- bound dye >100
fold increase fluorescence
TaqM an -- Hydrolysis Probe
M onitor acceptor fluorescence
hn
Hybridization probes
FRET
hn
donor acceptor
hn
fluor quencher
hn
Extension continues
Figure 2. Figure X. Schematic of SYBR Green I, TaqMan, and hybridization probe
Labelling approaches
CYBR green
Real-Time PCR
This technique allows quantitation of DNA
and RNA. Reactions are characterized by
the point in time during cycling when
amplification of a PCR product is first
detected rather than the amount of PCR
product accumulated after a fixed number
of cycles. The higher the starting copy
number of the nucleic acid target, the
sooner a significant increase in
fluorescence is observed.
TAQ-man probes
FRET probes

44. PCR fingerprinting
AP-PCR (arbitrarily primed PCR), 1 primer required, 10-20 bp, no sequence
information required
REP-PCR (repetitive extragenic palindromic sequences) 2 primers insert
randomly into the REP sites
ERIC-PCR (enterobacterial repetitive intergenic consensus sequences), 2
primers insert randomly into the ERIC sites, best for Gram Negative microbes
All of these fingerprinting techniques tell one if two isolates are the same or
different. They do not provide information about the identity or relatedness
of the organisms

45. RT-PCR lab
You have a cell…is a certain gene on
(by “on,” we mean active and
producing mRNA?)?
If a certain gene is on when the cell
divides, the gene might produce a
protein that causes cell division….

46. Central Dogma:
• DNA has genes and is in nucleus
• TRANSCRIPTION: Double strands of DNA unwind
to allow synthesis of messenger RNA (mRNA)
from one strand (the coding strand)
• The mRNA moves out of the nucleus to the
cytoplasm
• mRNA binds to Ribosomes to code for a protein-
protein made (translation)
• Protein carries out intent of gene (red hair
protein = hair gene)

47. DNA Structure

48. Unwind, mRNA is
made off DNA
template- similar to
this picture of DNA
made off of DNA.
Nucleotides pair up:
G always pairs with C,
T pairs with A. Except
in RNA, T is replaced
with U.

49. Transcription:
RNA synthesis
(note coding and
template strands)
(ch.21)

50. Making mRNA off DNA:

51. So, first step of RT PCR is:
• ISOLATE THE mRNA from the cell
• Next, make DNA from the mRNA
• This is reversing “transcription”– so use an
enzyme originally obtained from viruses–
ENZYME IS CALLED REVERSE TRANSCRIPTASE
(THE RT OF RT PCR)

52. • Last slide: this is the RT part of RT PCR
PCR part:
• After RT, you now have a tiny, trace amount
of what is called complimentary DNA (cDNA).
This tiny trace amount is not enough to
sequence.
• Next, you have to make enough copies of the
tiny trace amount of cDNA to sequence