Recombinant DNA Technology
Aug 22, 2024
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Cloning
Requirements of Cloning or Replication
Types of Cloning
Restriction Endonucleases
Hybridization or Blotting Technique
Western Blot
Qualities of Vector
Frequently Asked Questions
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
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:
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Sticky end Restriction Endonucleases |
Blunt end Restriction Endonucleases |
Ex: 3' G-G-A-T-C-C 5' - cuts the sequence between G-G.
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Ex: 3' G-G-A-T-C-C 5' 5' C-C-T-A-G-G 3'
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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
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Electrophoresis The electrophoretic tank contains gel, and Wells is taken. ↓ All DNA fragments are placed in Wells. ↓ The top side is negatively charged, and the bottom is positively charged. ↓ Negatively charged DNA fragments move toward the positive electrode. ↓ Short fragments move faster than long fragments. Denaturation The DNA fragments are then denatured. ↓ The DNA fragments are double-stranded. ↓ To make them available to the probe, they are covered by denaturation to be single-stranded. ↓ Blotting The gel is blotted on nitrocellulose paper. ↓ Hybridization
The blotting paper is then dipped into a radiolabelled probe taken in a petri dish. ↓ The radiolabelled probe attaches with the complementary sequences. ↓ Hybridization takes place. ↓ Washing Take away the hybridized blotting paper. ↓ Wash out the excess unbound probe. ↓ Autoradiogram Expose the hybridized blotting paper to X-ray. ↓ The gene of interest with the probe creates a band. ↓ Fragments with a gene of interest are identified. |
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Southern Blotting |
Northern Blotting |
Western Blotting |
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Western Blot
We will read about western blot in detail as it is a really important, frequently asked question.
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Take the HIV antigens. ↓ Separated by electrophoresis. ↓ Collect the patient's serum, which may or may not have antibodies. ↓ Add the serum to separate HIV antigens. ↓ If antibodies are present in the patient's serum, they hybridize with the antigen. ↓ Wash the unbound proteins. ↓ Add a labeled probe to the Ag-Ab complex, which is directed against the human antibody. ↓ 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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