All questions of Biotechnology: Principle & Processes for NEET Exam

Insertional inactivation is a technique used in molecular biology to identify recombinant clones. It involves the insertion of a foreign DNA fragment into a plasmid vector, which disrupts the function of a selectable marker gene. This results in the loss of the ability of the host cell to grow on a selective medium containing the corresponding antibiotic.

Mechanism

The mechanism of insertional inactivation involves the insertion of a foreign DNA fragment into a plasmid vector, which disrupts the function of a selectable marker gene. The selectable marker gene is usually a gene that confers resistance to an antibiotic, such as ampicillin. The disruption of this gene results in the loss of the ability of the host cell to grow on a selective medium containing the corresponding antibiotic. This allows for the identification of recombinant clones that have taken up the plasmid vector with the foreign DNA fragment.

Applications

Insertional inactivation is commonly used in molecular biology to identify recombinant clones. It is particularly useful in the construction of cDNA libraries and the screening of genomic DNA libraries for specific genes. It can also be used to study gene expression and regulation, as well as for the production of recombinant proteins.

Conclusion

In conclusion, insertional inactivation is a useful technique in molecular biology for identifying recombinant clones. It involves the insertion of a foreign DNA fragment into a plasmid vector, which disrupts the function of a selectable marker gene. This results in the loss of the ability of the host cell to grow on a selective medium containing the corresponding antibiotic, allowing for the identification of recombinant clones that have taken up the plasmid vector with the foreign DNA fragment.

In general genes that confer resistance to various antibiotics are used as selective marker in cloning vectors.

For replication of DNA their is specific seq. like TATA BOX

The smaller the size the farther it moves hence depend only on the size

Downstream processing

For gene transfer into the host cell without using vector microparticles made of tungsten and gold coated with foregin DNA are bombarded into target cells at a very high velocity. This method is called biolistics or gene gun which is suitable for plants.So the correct option is 'gold or tungsten'.

DNA fingerprinting can resolve:

Identification of a person:
- DNA fingerprinting is a technique used to identify individuals based on their unique DNA profiles.
- Every person's DNA is unique, except for identical twins, making DNA fingerprinting a highly accurate method for identification.
- By comparing DNA samples from a crime scene, for example, with the DNA profiles of suspects, forensic scientists can determine the identity of the perpetrator.

Paternity dispute:
- DNA fingerprinting can also be used to resolve paternity disputes.
- By analyzing the DNA profiles of a child and potential fathers, it is possible to determine the biological father.
- This is done by comparing specific DNA markers between the child and the potential fathers.
- If the child shares common DNA markers with a potential father that are not present in the other potential fathers, it indicates that the tested individual is the biological father.

Maternity dispute:
- Similarly, DNA fingerprinting can also be used to resolve maternity disputes.
- By analyzing DNA profiles of a child and potential mothers, it is possible to determine the biological mother.
- This is done by comparing specific DNA markers between the child and the potential mothers.
- If the child shares common DNA markers with a potential mother that are not present in the other potential mothers, it indicates that the tested individual is the biological mother.

All of the above:
- DNA fingerprinting can resolve all the mentioned scenarios: identification of a person, paternity disputes, and maternity disputes.
- This technique has revolutionized forensic science, allowing accurate identification of individuals involved in criminal activities.
- It has also played a crucial role in resolving legal disputes regarding parentage, ensuring the correct determination of biological relationships.

In conclusion, DNA fingerprinting is a powerful tool that can be used to resolve various scenarios, including the identification of individuals, determination of paternity, and resolution of maternity disputes. Its accuracy and reliability make it an indispensable technique in forensic science and legal proceedings.

Visualizing DNA Molecules Separated by Agarose Gel Electrophoresis

Agarose gel electrophoresis is a widely used technique to separate DNA molecules based on their size. The separated DNA fragments can be visualized using different methods.

Ethidium Bromide Staining and UV Light

Ethidium bromide is a commonly used fluorescent dye that intercalates into the DNA double helix and emits fluorescence under UV light. When DNA is stained with ethidium bromide and exposed to UV light, the dye binds to the DNA and fluoresces, allowing the DNA bands to be visualized.

Correct Statement

Option 'A' is correct in the context of visualizing DNA molecules separated by agarose gel electrophoresis. Ethidium bromide stained DNA can be seen under exposure to UV light. The other options are incorrect because DNA cannot be seen without staining in visible light, and ethidium bromide stained DNA cannot be seen in visible light. However, DNA can be seen in visible light if it is stained with other dyes such as SYBR Green.

Significance

Visualization of DNA molecules separated by agarose gel electrophoresis is important in various molecular biology applications, including genomic DNA analysis, PCR product analysis, and DNA sequencing. Ethidium bromide staining and UV light visualization are the most commonly used methods due to their simplicity, sensitivity, and cost-effectiveness. However, the use of ethidium bromide has some safety concerns due to its mutagenic and carcinogenic properties. Therefore, alternative DNA stains such as SYBR Green and GelRed have been developed, which are safer and more environmentally friendly.

Restriction endonuclease is a type of enzyme that can cleave molecules of DNA at a particular site called restriction site having polindromic sequence. These enzymes are produced by many bacteria and protect the cell by cleaving and destroying the DNA of invading viruses. Now a days, restriction enzymes are widely used in the techniques of genetic engineering.Therefore, the correct answer is option 'A'.

Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector.

• Log phase (or exponential phase) is actually characterized by a rapid and significant increase in the number of cells. During this phase, cells are dividing at an exponential rate.
• Lag phase is the initial phase where cells are adapting to new conditions and preparing for growth. During this phase, there is little to no significant increase in cell number as the cells are not yet dividing rapidly.

The suitable temperature for heat-shock treatment can vary depending on the specific experiment or protocol being used. However, a common temperature range for heat-shock treatment is typically between 37°C (98.6°F) and 42°C (107.6°F).

Overview of Restriction Endonucleases
Restriction endonucleases, also known as restriction enzymes, are crucial in molecular biology for manipulating DNA. Let's evaluate the statements provided regarding these enzymes.
Statement Analysis
- i. Hind II was the first restriction endonuclease discovered and cuts DNA at a specific sequence of six base pairs.
- This statement is incorrect. Hind II was not the first restriction enzyme discovered; that title goes to Hind II's predecessor, Hind I. Additionally, while Hind II does cut DNA at a specific site, it does not necessarily cut at a sequence of six base pairs.
- ii. The convention for naming these enzymes is the first 2 letters of the name comes from the genus and the second one letter comes from the species of the prokaryotic cell from which they were isolated.
- This statement is correct. For example, EcoRI is derived from Escherichia coli (genus: Eco, species: R).
- iii. Exonucleases and endonucleases are both types of restriction enzymes that function by cutting DNA at specific sites.
- This statement is incorrect. Exonucleases remove nucleotides from the ends of DNA, while endonucleases cut within the DNA strand. Only endonucleases are classified as restriction enzymes.
- iv. Each restriction endonuclease can only recognize and cut palindromic sequences in the DNA.
- This statement is correct. Most restriction enzymes recognize specific palindromic sequences in the DNA, which are sequences that read the same forwards and backwards.
Correct Statements
Based on the analysis, the correct statements are ii and iv. Thus, the correct answer is option 'c' (i and iv).

Purpose of treating bacterial cells or plant/animal tissue with enzymes like lysozyme, cellulase, or chitinase in the process of recombinant DNA technology:

Recombinant DNA technology involves the manipulation of DNA molecules to produce desired genetic combinations. This technology is widely used in various fields such as medicine, agriculture, and biotechnology. One of the crucial steps in recombinant DNA technology is the extraction of DNA from cells or tissues. Enzymes like lysozyme, cellulase, or chitinase are used in this process to achieve the following purposes:

To release DNA from cells:
The primary purpose of treating bacterial cells or plant/animal tissue with enzymes is to release DNA from the cells. These enzymes break down the cell walls or cell membranes, allowing the DNA to be isolated. Each of the mentioned enzymes has a specific role in this process:

1. Lysozyme: Lysozyme is an enzyme that breaks down the peptidoglycan layer of bacterial cell walls. By degrading the cell wall, lysozyme weakens the structure and facilitates the release of DNA from bacterial cells.

2. Cellulase: Cellulase is an enzyme that hydrolyzes cellulose, a major component of plant cell walls. By breaking down the cellulose fibers, cellulase helps in releasing DNA from plant tissues.

3. Chitinase: Chitinase is an enzyme that breaks down chitin, a substance found in the cell walls of fungi and some animal tissues. By degrading chitin, chitinase aids in the release of DNA from fungal or animal cells.

Other purposes:
Apart from releasing DNA from cells, these enzymes may also have additional roles in the process of recombinant DNA technology:

- Removal of proteins: Enzymes like lysozyme, cellulase, or chitinase can also help in removing proteins that may be present along with DNA. Proteins can interfere with the subsequent steps of DNA manipulation and analysis, so their removal is essential.

- Isolation of RNA: In some cases, these enzymes can be used to isolate RNA from cells or tissues. By breaking down the cell walls or membranes, they release both DNA and RNA, allowing for the isolation of RNA molecules.

- Precipitation of DNA: Although not directly related to the mentioned enzymes, the precipitation of DNA with ethanol is a common step in DNA extraction. After the release of DNA from cells, it is often necessary to concentrate and purify the DNA by precipitating it with ethanol.

In summary, the purpose of treating bacterial cells or plant/animal tissue with enzymes like lysozyme, cellulase, or chitinase in the process of recombinant DNA technology is primarily to release DNA from cells. These enzymes break down the cell walls or membranes, enabling the isolation of DNA for further manipulation and analysis. Additionally, they may assist in removing proteins, isolating RNA, or precipitating DNA during the extraction process.

Availability of thermostable dna polymerase made the rxn catalyed even at high temperature