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Recombinant DNA Technology

Aug 22, 2024

There are various parts of DNA recombinant technology. In this blog, we will read about them in detail. This is a very important topic in biochemistry, and many competitive exam questions can be expected from this topic.

Cloning

Cloning is the Production of multiple identical copies of DNA. It involves genetic engineering and has forensic applications as well. It refers to the replication of DNA multiple times.

Requirements of Cloning or Replication

The following things are required for cloning or replication.

DNA polymerase
Primer
All 4 deoxy nucleoside diphosphate (dNTPs)
Magnesium or manganese - acts as a catalyst
Buffer

Types of Cloning

Cell-Based Cloning	Enzyme Based Cloning
A whole cell, like E. coli, meets all the requirements. Introduce desired DNA fragments in E-coli. In vivo cloning. Includes recombinant DNA technology (rDNA).	A test tube is taken, and all the requirements are added to it. Cloning occurs in the test tube. In vitro cloning. Includes polymerase chain reaction (PCR).

To read about the Steps of Recombinant DNA Technology and the Rate-Limiting Step, sign up for the prepladder app and learn from the best medical faculties in India.

Restriction Endonucleases

Restriction Endonucleases cut the DNA at specific sites called palindromic sequences. Palindromic sequences are sequences that have the same pronunciation when read from both sides. For Example, the word ROTOR is a palindrome. The double-stranded sequence is the 5' to 3' sequence read in the reverse direction. For Example: 3' G-G-A-T-C-C 5' and 5' C-C-T-A-G-G 3'

The sequence from 3' to 5' on the first strand is the same as the second strand's when read from 5' to 3'. The two types of restriction endonucleases:

Sticky end RE
Blunt end RE

The following table differentiates the two types of restriction endonucleases:

Sticky end Restriction Endonucleases

Blunt end Restriction Endonucleases

Cut the DNA strands at different sites on the two strands.

Ex: 3' G-G-A-T-C-C 5' - cuts the sequence between G-G.

5' C-C-T-A-G-G 3' - cuts the sequence between G-G.
Forms two complexes with overhanging ends.
The gene of interest having a sticky end combines with a similar vector having a sticky end.

Cut both strands at the same site.

Ex: 3' G-G-A-T-C-C 5'

5' C-C-T-A-G-G 3'

Cuts the strands between A-T
From two complexes with blunt ends.
When genes of interest and vectors are brought together, they don't stick easily.
It is difficult to form a recombinant vector.

Today, mostly sticky end restriction endonucleases are used. The only blunt end restriction endonuclease used is Hpal.

Hybridization or Blotting Technique

Hybridization and blotting techniques involve many steps. These steps are enlisted as follows:

Electrophoretic separation
Denaturation of separated fragments
Blotting
Hybridization
Washing
Autoradiogram

Electrophoresis

The electrophoretic tank contains gel, and Wells is taken.

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All DNA fragments are placed in Wells.

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The top side is negatively charged, and the bottom is positively charged.

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Negatively charged DNA fragments move toward the positive electrode.

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Short fragments move faster than long fragments.

Denaturation

The DNA fragments are then denatured.

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The DNA fragments are double-stranded.

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To make them available to the probe, they are covered by denaturation to be single-stranded.

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Blotting

The gel is blotted on nitrocellulose paper.

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Hybridization

The blotting paper is then dipped into a radiolabelled probe taken in a petri dish.

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The radiolabelled probe attaches with the complementary sequences.

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Hybridization takes place.

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Washing

Take away the hybridized blotting paper.

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Wash out the excess unbound probe.

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Autoradiogram

Expose the hybridized blotting paper to X-ray.

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The gene of interest with the probe creates a band.

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Fragments with a gene of interest are identified.

Southern Blotting	Northern Blotting	Western Blotting
Used to identify an unknown DNA fragment. Denaturation is required to convert dsDNA to ssDNA. Labeled DNA is used as a probe.	Used to identify an unknown RNA sample. RNA is single-stranded. Mild denaturation is required to remove the hairpin loops. Labeled DNA is used as a probe.	Used to identify unknown proteins. An antibody probe is used. Confirmatory test for HIV.

Western Blot

We will read about western blot in detail as it is a really important, frequently asked question.

Take the HIV antigens.

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Separated by electrophoresis.

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Collect the patient's serum, which may or may not have antibodies.

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Add the serum to separate HIV antigens.

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If antibodies are present in the patient's serum, they hybridize with the antigen.

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Wash the unbound proteins.

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Add a labeled probe to the Ag-Ab complex, which is directed against the human antibody.

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If the labeled probe gives a signal, it indicates the presence of an antibody in the patient's serum.

Qualities of Vector

A vector should be capable of self-replication. It should have one or more origins of replication and a particular or desired restriction site.

A vector should have one or more marker genes to identify E-coli colonies with recombinant vectors. If natural vectors are used, like plasmids or phage DNA, choose them if they have:

Ampicillin resistance gene
Tetracycline resistance gene

The most commonly used marker genes are BAC or YAC. Artificial marker genes used are Beta-galactosidase.

Frequently Asked Questions

Q. Which of the following is a cell-based cloning technique?

a. Polymerase Chain Reaction

b. Restriction Fragment Length Polymorphism

c. Microarray

d. Recombinant DNA technology

Answer: D. Recombinant DNA technology

Q. All the following are the tools required for recombinant DNA technology except.

a. Restriction endonucleases

b. Blotting

c. Thermocycler

d. Vector

Answer: c. Thermocycler

Q. What is true about recombinant DNA technology?

a. It requires Taq DNA polymerase.

b. It is an in vitro cloning process.

c. It requires a vector.

d. The equipment required is a thermocycler.

Answer: c. It requires a vector

Q. Which is not true about sticky end-producing restriction endonucleases?

a. They cut both strands

b. They cut at palindromic sequences

c. Hpal is an example

d. It favors the formation of recombinant vector

Answer: c. Hpal is an example

Q. The rate-limiting step of recombinant DNA technology is

a. Formation of recombinant vector

b. Introduction of the recombinant vector into a cell

c. Identification of the fragment with a gene of interest

d. Cloning of recombinant vector

Answer: b. Introduction of the recombinant vector into a cell

Q. Which of the following is not true about Northern Blot?

a. cDNA is the probe

b. Does not involve the denaturation step

c. Separates fragments based on length

d. The probe can be labeled with a radioactive substance

Answer: b. Does not involve the denaturation step

Q. The most commonly used plasmid vector is:

a. pBR322

b. pUC19

c. pCEV

d. pUC18

Answer: a. pBR322

Q. To produce Insulin on a large scale, what is introduced into E.Coli?

a. Insulin gene

b. Insulin mRNA

c. Insulin cDNA

d. Insulin tRNA

Answer: c. Insulin cDNA

Q. A 25-year-old man was scheduled for an emergency appendectomy. As a part of the preoperative screening, HIV testing was done, and his serum was found to be repeatedly reactive in enzyme Immunoassay. To confirm the HIV diagnosis, a method in which individual proteins of an HIV-1 lysate were separated according to size by polyacrylamide gel electrophoresis. The viral proteins were then transferred onto nitrocellulose paper and reacted with the patient's serum. An antihuman immunoglobulin G (IgG) antibody conjugated with an enzyme was used as a label. Name the method used and mention the analyte that is detected by this method.

a. Southern Blot, HIV DNA

b. Northern Blot, HIV RNA

c. Western Blot, HIV Antibody

d. Southern Blot, HIV RNA

Answer: c. Western blot, HIV Antibody

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