PCR

Ans. Replication of DNA in vitro

Ans. Radio-labeled DNA probe

Steps in PCR

PCR uses DNA polymerase to repetitively amlify targeted portion of DNA. Each cycle doubles the amout of DNA in the sample, leading to exponential increase with repeated cycles of amplification. Thus amplification after ‘n’ number of cycle in (2)”. Twenty cycles provide an amplification of 10⁶ (million) and 30 cycles of 109 (billion).

PCR occurs in following steps –

i)      Isolation of target DNA sequence :- 

ii)     Primers construction:- 

iii)    Denaturation of DNA :- 

iv)    Annealing of primers to single stranded DNA :- 

v)     Chain extension:- 

Thus following are required in PCR :- Target double stranded DNA, two specific primers, a thermostable DNA polymerase (Taq polymerase), deoxyribonucleotides (dNTP).

Ans. is ‘b’ i.e., Thermus acquaticus

PCR is a method of enzymatic amplification of a target sequence of DNA.

It is sensitive, selective (specific) and extremely rapid means of amplifying any desired sequence of double stranded DNA, which can be as short as 50-100 base pairs (bp) and as long as 10 kbp.

In PCR, the DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq).

Taq polymerase is used because it is thermostable, not denatured at a temperature upto 95°C (in PCR DNA is to be heated to 94°-95° C for separation of strands).

Ans. is ‘b^‘ i.e., Taq polymerase 

PCR is a method of enzymatic amplification of a target sequence of DNAe.

• It is sensitive, selective (specific) and extremely rapid means of amplifying any desired sequence of double stranded DNA^e, which can be as short as 50-100 base pairs (bp) and as long as 10 kbp.
• In PCR, the DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq). Taq polymerase is use because it is thermostable⁰, not denatured at a temperature upto 95°C (in PCR DNA is to be heated to 94°-95° C for separation of strands).
• For amplifying a desired DNA sequence in DNA, we have to know short flanking sequences on either side of the target segue nce so that complementary primers can be prepared.
• Primers⁰ are the synthetic oligonucleotides of 20-35 sequence, which have sequence complementary to flanking sequence, i.e. sequence of flanking region of target DNA sequence.
• Primers are amplified to produce desired sequence of DNA.

Ans. (d) To know how much amplification of DNA has occurred 

Real-Time PCR

It is the molecular detection technique that discriminates real-time amplification from conventional PCR assays.

The real-time polymerase chain reaction (PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction.

This combines the DNA amplification and detection steps into one homogeneous assay and obviates the requirement for gel electrophoresis to detect amplification products.

Its simplicity, specificity, and sensitivity, together with its, more reliable instrumentation, and improved protocols, has made real-time PCR a hallmark technology for amplification of DNA.

Real-time PCR is extremely useful in medical microbiology, with the greatest impact on virology.

Ans. is ‘c’ i.e., Transformation [Ref Lippincott’ _s 5thle p. 479-83; Harper 28^th/e p. 395] Steps in PCR

• Isolation of target DNA sequence→  Primer construction → Denaturation of DNA→ Annealing of primers to single stranded DNA→ Chain extension.

Ans. is ‘b’ i.e., PFU Polymerase; & ‘e’ i.e., T-7 polymerase [Ref Textbook of PCR by Mike McPherson]

• The use of high fidelity DNA polymerases in PCR is essential for reducing the introduction of amplification errors in PCR products.
• Several thermostable DNA polymerases with 3′ → 5′ exonuclease – dependent proofreading activity have been introduced for high.
• Pfu DNA polymerase → Derived from Pyrococcus fusarious.
• Pwo DNA polymerase → Isolated from Pyrococcus woesei.
• KOD HiFi DNA polymerase → Isolated from Thermococcus Kodakaraensis.
• T7 DNA polymerase.

Ans: a. Uses heat labile DNA polymerase,[Ref Harper 30th/458-59; Lippincott 6th/479-83, 5th/497-83; Chatterjea er Shinde7th/267-272]

• Specificity^Q is based on the use of two oligonucleotide primers that hybridize to complementary sequence on opposite strands of DNA & flank the target sequence Double stranded DNA can be disrupted by heat or high pH, giving rise to single stranded DNA. The single stranded DNA serves as a template for synthesis of a complementary strand by replicating enzymes, DNA polymerase.
• Early PCR reaction used an E. coli DNA polymerase that was destroyed by each heat denaturation cycle. Substitution of a heat-stable DNA polymerase (Taq polymeraseY from Thermus aquaticus, obviates this problem & has made possible automation of the reaction, since the polymerase reactions can be run at 70°C