Molecular Diagnosis of Infectious Diseases
Module: Concepts of conventional PCR
Dr. Banshi Sharma Senior Veterinary Officer CVL
Polymerase Chain Reaction – an Introduction
Polymerase Chain Reaction (PCR) is the in vitro technique of enzymatically replicating DNA
Its purpose is to make large copy numbers of a gene for sequencing and/or detection
It requires:
• a template (or sample - DNA) • two primers
Primers
Short, artificial strands of DNA Determines PCR fragment Forward primer
DNA template
PCR fragment
Reverse primer
Taq Polymerase
Isolated from Thermus aquaticus Thermostable Assists in DNA replication
5’ 3’
5’
3’ 5’
3’
Thermocycler
Heating and cooling elements Contains thermal block and heated lid
Three step process
STEP 1: DENATURATION
Breaking of hydrogen bonds that connect the two strands of DNA DNA separates in two single strands
94°C
Three step process
STEP 2: ANNEALING
Primers attach to DNA strands
Temperature dependent on primer design
55 - 65°C
Three step process
STEP 3: EXTENSION
Copying of DNA strands by the DNA
polymerase
68 - 72°C
Conventional PCR
Conventional PCR
“Plateau effect” in PCR amplification
Actual PCR Product
DNA amount
Conventional PCR
Is there a gene copied during PCR and is it the right size ?
Before the PCR product is used in further applications, it has to be checked if :
a product formed
Gel Electrophoresis
Visualisation of PCR products
The ladder is a mixture of fragments with known size to compare with the PCR fragments. Lane 1 : PCR fragment is approximately 1850 bases
RNA viruses and PCR
Need to first make complimentary DNA (cDNA)
cDNA is made from mRNA An RNA dependent DNA polymerases operatesu onc single cstranded RNA to tg tcgug ttucg generates cDNA on pairing mRNA bases (A U RNA G First to DNART enzyme( T A C G ) C) step bases Creation of cDNA allows PCR in the traditional manner g a a t g performed t a c g a g c t c to be c
cDNA
Second step Traditional PCR
One step
Methods
Preparing reaction mixes
Resuspension of primers
Nucleic Acid Extraction Methods
RNA, DNA, manual, robotics
cDNA Synthesis Conventional PCR
two step RT-PCR
one step RT-PCR
Agarose gel electrophoresis
Preparation, running and documentation
Gel band purification