Gene Technology

Question 1

What is gene technology?

Answer 1

Gene technology refers to the modification or manipulation of an organism’s gene using technology.

Question 2

What are the different ways that genes can be extracted using gene technology?

Answer 2

1. A DNA fragment
2. Removed from a cell by cutting with restriction endonuclease
3. From a DNA library (BACs library)
4. From mRNA and reverse transcriptase
5. Making a gene: synthetic DNA

Question 3

What is mRNA, and how can it be used in gene technology?

Answer 3

1. mRNA carries genetic information and is read by ribosomes during translation
2. Extracted mRNA can be treated with reverse transcriptase enzyme
3. Reverse transcriptase enzyme creates single-stranded cDNA molecules with complementary base sequence to mRNA.
4. Obtained cDNA molecules are treated with DNA polymerase in PCR
5. DNA polymerase synthesizes a complementary DNA strand, creating double-stranded DNA
6. DNA molecules are replicated in repeated cycles to amplify the number of DNA fragments

Question 4

What are the advantages and disadvantages of using mRNA and reverse transcriptase in gene technology?

Answer 4

Advantages:
More copies are produced.
The process takes place in the cytoplasm.
Disadvantages:
The mRNA has to be converted to DNA first.

Question 5

What is PCR, and what are its main applications?

Answer 5

PCR (Polymerase Chain Reaction) is a cloning DNA method.
main applications include:
amplifying DNA for forensic tests and increasing the number of copies of a specific DNA sequence,

Question 6

What are the four components needed for the PCR process?

Answer 6

1. DNA sample - template DNA
2. DNA primers - Primers bind to the beginnings of the two DNA strands and allow the recruitment of polymerase.
3. DNA polymerase - enzyme replicates DNA fragments.
4. DNA nucleotides - building blocks for the synthesis of new DNA strands.

Question 7

What are the steps of PCR?

Answer 7

Step 1 - Denaturation: The master mix, primers, and DNA to be amplified are heated to at least 94°C, breaking the hydrogen bonds separating the DNA into single strands
Step 2 - Annealing: The mixture is then cooled to between 50 to 60°C, allowing DNA primers and DNA polymerase to bind to the strands of DNA.
Step 3 - Extending: The optimum temperature for Taq DNA polymerase is 72°C. Taq DNA polymerase uses free DNA nucleotides to replicate strands. Complementary DNA nucleotides are added to the primer.
At the end of each cycle, the number of DNA fragments doubles in size. After the first cycle, there are two copies of the original fragment.

Question 8

What is the process for inserting genes into vectors?

Answer 8

Restriction enzymes cut out the DNA of interest and open the vector. DNA ligase joins the DNA sequences together.

Question 9

What are marker genes?

Answer 9

Marker genes are used to determine whether the desired DNA has been taken up by the plasmid.

Question 10

What are reporter genes?

Answer 10

Reporter genes are genes that enable the detection or measurement of the inserted gene expression

Question 11

What is DNA sequencing?

Answer 11

DNA sequencing is a laboratory technique used to determine the precise order of nucleotides, or bases, in a DNA molecule.

Question 12

What is high-throughput sequencing?

Answer 12

1. High-throughput sequencing is a DNA sequencing technique that uses the chain-termination method.
2. In this method, each type of dideoxynucleotide is labeled with a specific fluorescent dye.
3. The single-stranded DNA chains are separated according to mass using capillary electrophoresis,
4. a laser beam is used to illuminate all of the dideoxynucleotides.
5. A detector then reads the color and position of each fluorescence, and the information is fed into a computer for storage or analysis.

Question 13

What is CRISPR?

Answer 13

1. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a natural defensive mechanism used by bacteria to fight against viral infections.
2. The CRISPR-Cas9 system is made up of redundant repeat regions and variable spacer regions.
3. Spacer regions are short segments of DNA taken from viral invaders, and the long stretch of repeats and spacers is called a “CRISPR array.”

Question 14

What are the steps of the bacterial immune response (of CRISPR)?

Answer 14

1. Expression: the CRISPR array is transcribed into a long precursor CRISPR RNA molecule, which is then cleaved into small CRISPR RNAs (crRNAs)
2. Interference: a crRNA and a second RNA (tracrRNA- trans-activating CRISPR RNA) form a complex with Cas9 a bacterial enzyme.
3. Scanning: The Cas9-RNA complex scans along DNA randomly; if it finds a sequence that matches the crRNA, it stops and then cuts the target DNA. The DNA is then repaired by the cell
4. The Cas9 enzyme has been turned into a programmable genome editing tool

Question 15

What are the uses of CRISPR?

Answer 15

CRISPR has several uses:
1. developing new varieties of plants with drought resistance, pest resistance
2. DNA base editing involves tethering dCas9 to an enzyme that can convert a single, specific nucleotide base into another (e.g. A into a G).
3. CRISPR can be used to knock out genes, producing cell lines with random deletions around a specific sequence,

Question 16

Question: What is gel electrophoresis?

Answer 16

A technique used to separate charged molecules like DNA, RNA, and proteins according to their size.
Commonly used in laboratories.

Question 17

Question: What are the steps involved in gel electrophoresis?

Answer 17

1. DNA sample loading with several fragments of different sizes and concentrations.
2. Application of electrical field using positive and negative electrodes at opposite ends of the gel.
3. DNA separation through the migration of macromolecules towards the opposite charge.
4. UV transillumination and documentation to visualize the stained macromolecules.

Question 18

Question: What is DNA Southern Blotting?

Answer 18

A technique used to examine nucleic acid samples for certain sequences.

Question 19

What are the steps involved in DNA Southern blotting?

Answer 19

1. Uses gel electrophoresis to separate nucleic acid fragments by size.
2. Transfers fragments to a nylon membrane through blotting.
3. Binds probe nucleic acid fragments to the membrane surface using specific radioactively or fluorescently labeled probe sequences.
4. Visualizes any DNA fragments containing complementary sequences with the DNA probe sequence within the Southern blot.