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प्रश्न
What does PCR stand for? Describe the different steps of PCR
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उत्तर
PCR stands for Polymerase Chain Reaction:
Basic requirements for PCR technique are :
1) A DNA segment (100-35, 000 bp in length) be amplified.
2) Primers (forward and reverse) which are synthetic oligonucleotides of 17-30 nucleotide. They
are complementary to the sequence present on the desired DNA segment.
3) Four types of deoxyribonucleotides (dATP, dCTP, dGTP, dTTP). They are collectively called
dNTPs
4)A thermostable DNA polymerase, that can withstand up to 94°C. Usually, Taq polymerase isolated from bacterium Thermus aquaticus is used.
The three essential steps for PCR technique are :
1) Heat denaturation: This step involves heating of DNA at about 91°C. The heating breaks the hydrogen bonds to make ssDNA. The DNA molecule with more G-C pairs needs the higher
temperature.
2)Annealing: It is pairing of primers to the ssDNA segment. The primer has to be designed as per the requirement. this step requires temperature at about 55°C.
3) Polymerisation: The temperature is raised to 72°C. The Taq polymerase adds dNTPs behind
the primer on the ssDNA
These three steps constitute one cycle of the reaction. The process is carried out for about 28-30 cycles beyond which its reliability decreases.
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संबंधित प्रश्न
Explain what are the desirable characteristics of an ideal cloning vector used in rDNA technology.
Why are bio-fertilizers preferred over chemical fertilizers?
Which of the following is TRUE regarding EcoR I?
The following identifies the major features of technology that differentiate modem biotechnology from classical biotechnology.
i. Capability of science to change the genetic material for getting new specific products through rDNA technology, polymerase chain reaction (PCR), microarrays, cell culture and fusion, and bioprocessing.
ii. Ownership of technology and its sociopolitical impact.
iii. Modem biotechnology makes use of only fermentation technology.
iv. Classical biotechnology relies only on principles of plant and animal tissue culture.
In which of the following techniques, charged molecules (DNA, RNA and Proteins) are separated by applying the electric field?
Exonuclease performs the function of ____________.
Restriction enzymes were discovered by ______.
Which of the following statements are true about cloning vectors?
- Vectors are DNA molecules that carry a foreign DNA segment.
- The replicated DNA is introduced into the host cell.
- It possesses the origin of replication (Ori).
- Vectors may be plasmid, bacteriophage, Yeast artificial chromosomes (YACs), etc.
- Vectors do not require a selectable marker to identify and eliminate non-transformants.
Choose the correct answer from the options given below.
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.
______technique is used for in vitro gene cloning or gene multiplication.
