PCR is a laboratory technique used to amplify a specific region of DNA through multiple cycles of heating and cooling. It allows a small amount of DNA to be exponentially amplified into millions of copies. The key components of PCR are DNA template, primers, DNA polymerase, and nucleotides. During each cycle, the DNA is denatured, primers anneal to the DNA, and the polymerase extends the primers to synthesize new DNA strands. This allows the specific target sequence to be rapidly amplified. There are various types of PCR that have different applications in research, forensics, and medicine.

1. Polymerase Chain Reaction

2. Introduction
• PCR, polymerase chain reaction, is laboratory
technique (in-vitro) for amplification of a region
of DNA whose sequence is known or which lies
between two regions of known sequence
• PCR use smallest sample of the DNA to be cloned
and amplify it to millions of copies- few hours
• Before PCR, DNA of interest could only be
amplified by over-expression in cells--- with
limited yield

3. • 1983, Kary Mullis postulated the concept of
PCR ( Nobel Prize in 1993)
• PCR become essential and routine tool in
most biological laboratories

4. Principle of PCR
• PCR involves primer mediated enzymatic
amplification of DNA
• based on using the ability of DNA polymerase to
synthesize new strand of DNA complementary to
template strand
• Primer needed b/c DNA polymerase add a
nucleotide only onto a preexisting 3′-OH group to
add the first nucleotide
• DNA polymerase then elongate 3 end by adding
more nucleotides to generate an extended region
of double stranded DNA

5. Components of PCR
• DNA Template: The double stranded DNA (dsDNA) of
interest, separated from the sample (genomic)
• DNA Polymerase: thermostable Taq polymerase not
rapidly denature at high temperatures (98°), function
temperature optimum of about 70°C
• Oligonucleotide primers: Short pieces of single stranded
DNA (often 20-30 base pairs) which are complementary to
the 3’ ends of sense and anti-sense strands- target sequence
• Deoxynucleotide triphosphates: Single units of the bases
A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the
energy for polymerization and the building blocks for
DNA synthesis
• Buffer system: magnesium and potassium to provide
optimal conditions for DNA denaturation and renaturation;
also important for polymerase activity, stability

6. 1- DNA template
• DNA containing
region to be
sequenced
• Size of target DNA
to be amplified : up
to 3 Kb

7. 2- Primers
• 2 sets of primers
(Forward, Reverse)
• Generally 20-30
nucleotides long
• Synthetically
produced
• complimentary to the
3’ ends of target DNA

8. Primers
• Not containing inverted repeat sequences to
avoid formation of internal structures
• 40-60% GC content preferred for better
annealing

9. 3-Enzyme
• Usually Taq Polymerase or anyone of the
natural or Recombinant thermostable
polymerases
• Stable at T0 up to 950 C
• High processivity

10. Procedure of PCR
• All PCR components-mixed together- taken through series
of 3 major cyclic reactions conducted in automated, self-
contained thermocycler machine
• Denaturation:
heating- reaction mixture to 94°C for 15-30 seconds
double stranded DNA- denatured to single strands due to
breakage in weak hydrogen bonds
• Annealing:
reaction temperature-rapidly lowered to 50-60°C for 20-40
seconds -allows primers to bind (anneal) to their
complementary sequence in the template DNA

11. • Elongation/Extension :
• usually occurs at 72-80°C, the polymerase enzyme in
sequence adds bases to 3′ each primer, extending the DNA
sequence in the 5′ to 3′ direction
• optimal conditions, DNA polymerase will add about 1,000
bp/minute
• With one cycle, a single segment of double-stranded DNA
template is amplified into two separate pieces of double-
stranded DNA
• These two pieces are then available for amplification in the
next cycle
• cycles are repeated, more and more copies are generated

17. Standard thermocycle

19. Types of PCR
• Real-time PCR
• Quantitative real time PCR (Q-RT PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Multiplex PCR
• Nested PCR
• Long-range PCR
• Single-cell PCR
• Fast-cycling PCR
• Methylation-specific PCR (MSP)
• Hot start PCR
• High-fidelity PCR
• In situ PCR

20. • Variable Number of Tandem Repeats (VNTR) PCR
• Asymmetric PCR
• Repetitive sequence-based PCR
• Overlap extension PCR
• Assemble PCR
• Intersequence-specific PCR(ISSR)
• Ligation-mediated PCR
• Methylation –specific PCR
• Miniprimer PCR
• Solid phase PCR
• Touch down PCR, etc

21. Applications of PCR
• Medical Applications:
• Genetic testing for presence of genetic disease mutations
(hemoglobinopathies, cystic fibrosis, other inborn errors of
metabolism)
• Detection of disease causing genes in suspected parents
who act as carriers
• Study of alteration to oncogenes may help in customization
of therapy
• be used as part of a sensitive test for tissue typing, vital to
organ transplantation
• genotyping of embryo
• monitor the gene in gene therapy

22. • Infectious disease Applications:
• Analyzing clinical specimens for the presence of infectious
agents, including HIV, hepatitis, malaria, tuberulosis etc.
• Detection of new virulent subtypes of organism that is
responsible for epidemics
• Forensic Applications:
• be used as a tool in genetic fingerprinting
• can identify any one person from millions of others in case
of : crime scence, rule out suspects during police
investigation, paternity testing even in case of availibility
of very small amount of specimens ( stains of blood,
semen, hair etc)

23. • Research and Molecular Genetics:
• In genomic studies:
• compare the genomes of two organisms and identify the
difference between them
• In phylogenetic analysis.
• Minute quantities of DNA from any source such a
fossilized material, hair, bones, mummified tissues
• In study of gene expression analysis, PCR based
mutagenesis
• In Human genome project for aim to complete mapping
and understanding of all genes of human beings

24. Types of PCR
• Conventional PCR
• Multiplex PCR
• Nested PCR
• RT PCR and qRT PCR
• Quantitative PCR
• Hot Start PCR
• Touch Down PCR
• Assembly PCR
• Colony PCR
• Methylation Specific PCR
• LAMP Assay

25. Multiplex PCR
• uses several pairs of primers annealing to different target
sequences
• permits the simultaneous analysis of multiple targets in a
single sample
• in testing for genetic mutations, six or more amplifications
might be combined
• standard protocol for DNA Fingerprinting, the targets
assayed are often amplified in groups of 3 or 4

26. Nested PCR
• 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 may contain products amplified
from non-target areas
• The products from the first PCR are then used as template
in a second PCR, using one ('hemi-nesting') or two
different primers whose binding sites are located (nested)
within the first set, thus increasing specificity
• Nested PCR- often more successful in specifically
amplifying long DNA products than conventional PCR
• but requires more detailed knowledge of the sequence of
the target

27. Quantitative PCR
• used to measure the specific amount of target DNA (or
RNA) in a sample
• 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

28. Real-Time PCR (QRT-PCR)
• Quantitative Real-Time PCR (QRT-PCR)
• use fluorescent dyes, such as Sybr Green, or fluorophore-
containing DNA probes, such as TaqMan, to measure the
amount of amplified product as the amplification
progresses

29. Hot-start PCR
• A technique performed manually by heating the reaction
components to the DNA melting temperature (e.g. 95 °C)
before adding the polymerase
• non-specific amplification at lower temperatures is
prevented
• Alternatively, specialized reagents inhibit the polymerase's
activity at ambient temperature, either by the binding of
an antibody, or by the presence of covalently bound
inhibitors that only dissociate after a high-temperature
activation step
• 'Hot-start/cold-finish PCR' is achieved with new hybrid
polymerases that are inactive at ambient temperature and
are only activated at elevated temperatures

30. Touchdown PCR
• The annealing temperature is gradually decreased in later
cycles
• The annealing temperature in the early cycles is usually 3–
5 °C above the standard Tm of the primers used, while in
the later cycles it is a similar amount below the Tm
• 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

31. Assembly PCR
• also known as Polymerase Cycling Assembly or PCA
• the synthesis of long DNA structures by performing PCR
on a pool of long oligonucleotides with short overlapping
segments, to assemble two or more pieces of DNA into
one piece
• involves an initial PCR with primers that have an overlap
and a second PCR using the products as the template that
generates the final full-length product
• This technique may substitute for ligation-based assembly

32. Colony PCR
• bacterial colonies are screened directly by PCR
• E.g the screen for correct DNA vector constructs
• Colonies are sampled with a sterile pipette tip and a small
quantity of cells transferred into a PCR mix
• To release the DNA from the cells, the PCR is either
started with an extended time at 95 °C
(when standard polymerase is used), or with a shortened
denaturation step at 100 °C and special chimeric DNA
polymerase

33. COLD-PCR
• co-amplification at lower denaturation temperature-PCR
• a modified protocol that enriches variant alleles from a
mixture of wild type and mutation-containing DNA

34. RT-PCR (Reverse Transcription PCR)
• used to reverse-transcribe and amplify RNA to cDNA
• PCR is preceded by a reaction using reverse transcriptase,
an enzyme that converts RNA into cDNA
• The two reactions may be combined in a tube with the
initial heating step of PCR being used to inactivate the
transcriptase
• polymerase has RT activity and can carry out the entire
reaction
• RT-PCR is widely used in expression profiling which
detects the expression of a gene

35. RT-PCR (Reverse Transcription PCR)
• can also be used to obtain sequence of an RNA transcript,
which may aid the determination of the transcription start
and termination sites and facilitate mapping of the location
of exons and introns in a gene sequence

36. Methylation-specific PCR (MSP)
• used to identify patterns of DNA methylation at cytosine-
guanine (CpG) islands in genomic DNA
• Target DNA is first treated with sodium bisulfite, which
converts unmethylated cytosine bases to uracil, which is
complementary to adenosine in PCR primers
• Two amplifications are then carried out on the bisulfite-
treated DNA
• One primer set anneals to DNA with cytosines
(corresponding to methylated cytosine) and the other set
anneals to DNA with uracil (corresponding to
unmethylated cytosine). MSP used in quantitative PCR
provides quantitative information about the methylation
state of a given CpG island.

37. LAMP Assay
• Loop mediated isothermal amplification
• Modified type of PCR using 3:6 –primers set
• One of them is loop like primer
• Used Bst polymease enzyme
• (Bacillus stearothermophilus DNA polymerase)
• Using only two tempreatures (63 ºC for 45 min. then 85 ºC
for 5 min.)
• May be carried out in water bath