Advertisements
Advertisements
प्रश्न
What is PCR? Explain different steps involved in it.
Advertisements
उत्तर
Definition: Polymerase Chain Reaction (PCR) is the process of in vitro amplification of gene of interest using a PCR machine.
Mechanism of PCR: At the start of PCR, all the requirements are mixed together in ‘eppendorf tube’ and the following operations are performed sequentially:
Step i: Denaturation
The reaction mixture is heated to a temperature (90–98o C) to separate two strands of desired DNA. This is called denaturation.
Step ii: Annealing
The mixture is allowed to cool (40–60o C) that permits pairing of the primer to the complementary sequences in DNA. This step is called annealing.
Step iii: Primer extension / Polymerization
The temperature (70–75o C) allows thermostable Taq DNA polymerase to use single-stranded DNA as template and adds nucleotides. This is called primer extension. It takes around two minutes duration.
संबंधित प्रश्न
What would be the molar concentration of human DNA in a human cell?
What is the cell that receives a recombinant gene called?
What is plasmid?
Observe the given sequence of nitrogenous bases on a DNA fragments and answer the following questions:
5' - CAGAATTCTTA - 3'
3' - GTCTTAAGAAT - 5'
- Name a restriction enzyme which can recognise this DNA sequence.
- Write the sequence after digestion.
- Why are the ends generated after digestion called sticky ends?
Which vector is mostly used in rDNA technology in plants?
pBR322, used very frequently in cloning experiments as a vector in E. coli is ______.
The structure below shows pUC18, which is similar to pBR322 in its function. However, they differ in some of their restriction sites and the number of ori. The ori number for pBR322 is approximately 20.
- How are puc18 and pBR322 used in biotechnological studies?
OR
What will be the impact if the ori in the above structure gets damaged? - The lac z gene has many recognition sites. Study the segment of DNA given below and answer the questions.
5’... ATC GTA AAG CTT CAT…3’
3’... TAG CAT TTC GAA GTA…5’
i) Applying your knowledge of palindrome sequences identify and mark the possible region where the restriction enzyme X will act.
ii) Restriction enzyme Y was used to extract a gene of interest from a plant. This gene needs to be inserted in the given DNA segment which has been treated with restriction enzyme X. Will there be a successful recombination? Explain with a reason. - Which one of the two (pUC18 and pBR322) would you prefer for biotechnological studies? Justify.
In vitro amplification of DNA segments is called______.
How many sets of primers are required in each cycle of PCR?
State the steps involved in the process of gene therapy for the treatment of ADA-deficiency.
