✓Standard PCR, DNA

✓RT-PCR, RNA

✓Real-Time PCR – RTQ-PCR

(DNA or RNA)

✓Digital PCR
Standard-PCR
 Nested-PCR

Human T Cell Lymphotropic Virus Type 2

Multiplex-PCR
 Random amplified polymorphic DNA

DNA is cut and amplified using short

single primers at low annealing
temperatures, resulting in
amplification at multiple loci. RAPDs
lack specificity, due to low annealing
temperatures and easier reaction
conditions.
 Long PCR

Long PCR used if the DNA

amplification up to 27 kb fragments.

The method relies on a mixture of :

Thermostable DNA
polymerases(Taq DNA)
Proofreading enzymes (e.g Pfu).
Restriction fragment length
polymorphisms (RFLP)

PCR-RFLP analysis and automated sequencing of MTHFR C667T. MTHFR was

restricted by HinfI. Digestion resulted in a 400-bp fragment for the C allele,
and 318 and 82 bp fragments for the T allele.
 Amplified fragment length
polymorphism (AFLP)

DNA is cut with two restriction enzymes to

generate specific sequences, which are
then amplified suitably.
The mere addition or deletion of bases at
the 3′ end determines the selectivity and
complexity of the amplification.
//----GAATTC---//----TTAA---// DNA sequence
//----CTTAAG--//-----AATT---// with EcoRI and
EcoRI MseI MseI recognition
sequences
---G AATTC---//----T TAA----// DNA restriction
---CTTAA G---//---AAT T
nnnA AATTC---//----- Addition of adaptors.
T TACnnnn As nucleotides in
nnnTTTAA G--//----- red are different to
AAT Gnnn the original ones
(blue), restriction
sites are not reconstructed
ASYMMETRIC PCR
 Direct sequencing and hybridization probing

 Amplifies just one strand of the target DNA

 First produce Double stranded DNA

 Then to produce single stranded DNA

 Unequal primer concentrations

 Amplification become slow after the one Primer used so Increase cycles

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 Steps

 DNA sample

 Add two primers of different

concentration
 Run PCR

 Result on Gel Electrophoresis

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 COLONY PCR
 Colony PCR for determining the presence or absence of insert
DNA in plasmid of Bacteria
 Size of the DNA sequence

 Biotechnology Products

 No need for Extraction and culturing of DNA or plasmid

purification steps

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 Steps

 Small quantities of bacterial cells from bacterial colonies are directly added

 Add desired Primers for amplification

 Run PCR

 Results on Gel Electrophoresis

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V A R I A T I O n S O F P C R :

PCRis highlyversatiletechniqueandhasbeenmodifiedin
varietyofwaytosuitspecificapplications.

 InversePCR
-In this methodamplificationofDNAofunknownsequenceis
carriedoutfromknownsequence.
- Thisis especially useful inidentifyingflankingsequencesof
various genomicinserts.

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