PCRPOLYMERASE CHAIN REACTION

Dr. Venkateswaran A.

PCR• Not a discovery – It is an Invention

• It is in one line – Making millions of copies of a DNA fragment

• Kary Mullis - inventor of pcr – won Nobel prize in 1993

• The foundation for this invention was built on the concept put forward by Dr. HarGobind Khurana in 1971

Thermal Cyclers

Thermal Cyclers

• Thermal Cyclers used

• Reactions in Micro titre plates or tubes

• Process in short is Cycles of Heating the reaction mixture

Ingredients of PCR

1 D/W

2 10X Buffer

3 MgCl2, EDTA

4 dNTPs

5 Primers

6 DNA Template

7 Taq

Ingredients of PCR

• 10X Buffer -- BSA, DMSO, Glyserol, Detergents, Gelatine – Allows Macromolecular crowding

• Mg ions 1.5 mM soln, K ions, (Mn ions)• dNTPs – 0.2 mM of each of the 4 dNTPs• Primers – 2 – a)complimentary to the 5’ end b)

complimentary to the 3’ end of the DNA fragment to be amplified.

• DNA fragment to amplified – The Templates

100- 3000 bases

upto 10000 bases. Errors more in longer fragments• Taq / Pfu / MuLV-RT

PCRMelting

94 oC

Melting

94 oC

AnnealingPrimers

50 oC

Extension

72 oCT

empe

ratu

re

100

0

50

T i m e

30x

5’3’

3’5’

3’5’

5’

5’3’5’

3’5’

5’

5’

5’

5’3’

3’5’

3’5’

5’3’

5’3’

5’

STEPS

• Initialisation – Heat to 95 degrees C – hold for 1-9 mins – in Hot-Start PCR

• Denaturation – Heat to 95 degres C and hold for 20-30 secs – Hydrogen bonds between the 2 strands broken and the dsDNA converted to ssDNA- Templates.

• Annealing – Temp brought down to 50 -65 degrees C (3-5 degrees below Tm of the Primer) hold for 20-30 secs. The enzyme binds the Primers to the complimentary sites in presence of Mg ions

STEPS• Extension / elongation-Heat to 72 degrees C (if

Taq is used)and hold for 30 secs. DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand

• 32- 35 Cycles of Denaturation, Annealing and Elongation

• Final elongation – Heat to 72 degrees C and hold for 12-15 mins to allow complete extension of all the amplicons

• Final hold – at 4 -15 degrees C for indefinite time for short time storage of the reaction

DNA Between The Primers Doubles With Each Thermal Cycle

0Cycles

Number1

3

8

2

4

1

2

4

16

5

32

6

64

Theoretical Yield Of PCRTheoretical yield = 2n-1x y

Where y = the starting number of copies and

n = the number of thermal cycles

= 107,374,182,400

If you start with 100 copies, how many copies are made in 30 cycles?

2n-1 x y

= 229 x 100

= 1,073,741,824 x 100

PCR in Simple Terms

An invisible quantity of few molecules of a DNA fragment yields millions of copies of the same DNA fragment in visible quantity in a short time

More Cycles = More DNAYield is confirmed by Gel Electrophoresis

Number of cycles 0 10 15 20 25 30

SizeMarker

Applications• 1) Gene Sequencing• 2) Molecular Diagnosis – Genetic Disorders,

Infectious Diseases eg. a) HIV virus before Ab are detectable, b) TB bacilli 1 per 1,00,000 human cells can be amplified and detected. c) Cancers – early detection certain Cancers. d)Mutations in growth cotrol genes (ras) for cancers. e) Monitoring Chemotherapy in Leukaemia – To stop treatment when abnormal DNA is eliminated – Ideal in Leukemias f) To detect recurrance of Cancers g) DNA in artificial knee / hip joints to detect infection

Applications• 3) Forensic Use –a) Paternity Testing b)

Identification at the time of Immigration c) Traces of DNA from sites of crime like assaults, murder, rape etc from dried blood, semen, saliva, single complete hair, DNA material of the accused from the inside of finger nail tips d) An individual’s Genetic profile is highly distinctive because many Genetic Loci are highly variable – DNA is highly stable if protected from water air and light and can remain intact indefinitely

Applications• Historians and other scientists – a) Russian

czars, b) 7,000 years old Egyptian Mummies, c) 40,000 years old Mammoth preserved in Ice in the Arctic d) to learn Evolutionary relationships between organisms, e) genes of microorganisms that cannot be cultured g) Mitochondrial DNA from various modern human populations indicating that Homo Sapiens originated in Africa supporting Fossil evidence

Applications• DNA from saliva, hair, skin, excreta of

organisms/animals/birds that cannot be caught. To estimate population size of animals / birds from their excreta in a given area. DNA in the GI Tract of Carnivores reveal Food web interactions. DNA in products made from endangered species like powdered Rhinocerus horn sold as aphrodisiac. DNA from food prepared from the meat of endangered species.

Denaturation: DNA meltsAnnealing: Primers bindExtension: DNA is replicated

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