PCRPolymerase Chain Reaction

Thermocycler.Typies & Applications

BY: AQEEL HADITHE osmania university BIOCHEMISTRY sem. III 1007-13-514-005

20-9-2014

( Very good mind behind

this techniques )

Kary Banks Mullis - 1983

a scientist working for the ( Cetus Corporation )

A Biotech Company of USA - northern California when he came up with the idea for the polymerase chain reaction. --------------------------------------------------------------------------------- Developed PCR in 1983

won the Nobel Prize in 1993.

Polymerase Chain Reaction• Polymerase chain reaction (PCR) :- Is nucleic acid amplification technology , that allows small amounts of genetic material to be amplified into billions of copies in just a few hours .

---( enzymatically replicating of DNA without using a living organism, such as E. coli or yeast )

--- The techniques was developed based on the discovery of the (biological activity at high temperatures of DNA polymerases ) Which found in thermo.philes (bacteria that live in hot springs).

Materials needed for PCR (Reaction Components)Reaction Components)

--------------------------------------------------------------------------------------

1- Target DNA (the DNA you want to copy)

( DNA region to be amplified )

The DNA can be from - animals - plants - viruses - bacteria.

Range concentration - 1-2 µl ( for a total reaction mixture of 10 µl)

2 - Free Nucleotides (A, T, C, G) :- PCR Nucleotide Mix is a premixed solution containing the sodium salts of (De.oxy nucleotide triphosphate ) dATP, dCTP, dGTP and dTTP, -- each at a concentration of 10mM in water. -- They are the building blocks from which the DNA polymerases synthesizes a new DNA strand.

Range concentration - 0.5 µl (for 10µl reaction mixture)

3- DNA Primers ( not RNA Primes ) -- Two primers ( forward & reverse ) -- short.long -20 nucleotides sd –DNA ( oligonucleotides ) that are synthesized to correspond to the ( beginning and ending ) of the DNA stretch to be copied .-- They are complementary to the 5' or 3' ends of the DNA region

-- Optimal length of PCR primers is ( 18-22 bp ) Range conc. - 1 µl ( for a total reaction mixture of 10 µl)------------------------------------------------------------------------------------------------------------------------------------

* We can also use RNA primer in PCR But There are two main reason why we are not using it :-

-- first point :- is ( tability ) DNA primer is more stable than RNA

-- second point :- is ( hybridi.zation ) DNA primer bind to template more efficient than RNA primer.

• Primers: Short artificial DNA sequences which define the DNA sequence to be amplified as they bind (anneal) to the DNA template and act as starting points for the DNA polymerase.

• - specificity and the temperature of annealing are ----- ( partly dependent )on primer length

• The primers should be ~20 bases ( long enough for adequate thespecificity ), and short enough to bind easily to the template at the annealing temperature.

• the primers should not be too short ----- ( as specificity decreases )

Primer length

Primer.. Secondary Structures : IN PCR we must avoid Cross homology --- Primers designed for a sequence must not amplify other genes in the mixture.

Primer Secondary Structures ---- produced by intermolecular interactions

• Lead to poor or no yield of the product.

Such as :-

– Hairpins intermolecular interaction within the

primer ( in the primer itself )

- Self Dimer intermolecular interactions

( between the two primers ),

- where the primer is homologous to itself.

They reduce the product yield.

4 - Taq Polymerase (heat stable DNA Polymerase III)

An enzyme that ( able to work in high Temo. 95 C )moves along the segment of DNA, reading its code and assembling a copy Range 0.2ul of (in 10µl of reaction mix)-------------------------------------------------------------------------------------------------------------------

5- Buffer solution•Contains Divalent cations like Mg+2 -- Mg2+ (cofactor that DNA Polymerase III needs to work) -- Provides suitable chemical environment for optimum activity and stability the DNA polymerase and other components of the reaction. Range - 1µl ( for a total reaction mixture of 10 µl)---------------------------------------------------------------------------------------------------------------------------------------------------------------------

6 - Sterile deionized water It’s quantity is variable--------------------------------------------------------------------------------------------------------------------------------------------------------------------------

7 - Thermocycle PCR machine (machine that changes temperatures)

PCR is repeated cycling of three steps:

1. Denature DNA

The DNA is heated to 95° C. This breaks the weak hydrogen bonds that hold DNA strands together in a helix, allowing the strands to separate creating single stranded DNA.

2. Primer Annealing

The mixture is cooled to 50° C. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.

3. Extension

The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template.

4. Go to Step 1 ( 20 - 35 X )

--------------------------------------------------------------------------------------

- These steps are repeated 20-35 times.

- In PCR, amplification is exponential because for each cycle, the DNA made in the previous cycles can also serve as template .

PCR

Melting for all the DNA

94 oClong time

Melting for only the fragment DNA

94 oCShort timeAnnealing

Primers

50 oC

Extension

72 oC

Tem

pera

ture

100

0

50

Time

30x

5’

Types of the PCR

• Conventional (basic) PCR • Restriction fragment length polymorphism (RFLP)PCR • Multiplex tandam PCR (MT-PCR)• Nested PCR• Random amplification of polymorphic DNA PCR (RAPD)• Amplified Fragment Length Polymorphisms (AFLPs ) PCR• Reverse Transcriptase PCR (RT-PCR)• Real time PCR (Rtime-PCR)• Colony PCR• Hot Start PCR• Asymmetric PCR• Long PCR• Allele specific PCR

Some of PCR application1 - DNA fingerprinting

2 - Production of DNA for sequencing

3 - Mapping the human genome

4 - The isolation of a particular gene

5 - Generation of probes

6 - Cloning a Gene encoding a known protein

7 - Amplification of ( old DNA - cloned DNA from Vectors )

8 - Detecting Bacterial or Viral Infection

a- AIDS infection

b- Tuberculosis (Mycobacterium tuberculosis) 9 - Genetics Diagnosis a - Diagnosing inherited disorders - Cystic fibrosis - Muscular dystrophy - Haemophilia A and B - Sickle cell anaemia b- Diagnosing cancer c- Blood group typing

http://www.mutationdiscovery.com/md/MD.com/screens/optimase/MasterMixCalculator.jsp?action=none