Recombinant DNA Technology

Question 1

Stages of Genetic Fingerprinting

Answer 1

1. Extraction
2. Digestion
3. Separation
4. Hybridisation
5. Development

Question 2

Extraction during genetic fingerprinting

Answer 2

DNA is extracted by phenol and chloroform, separating it from the rest of the cell.

The quantity of DNA is increased by the polymerase chain reaction

Question 3

Digestion during genetic fingerprinting

Answer 3

DNA is cut into fragments by restriction endonucleases.

Restriction endonucleases are chosen because they cut close to, but not within, the target DNA

Question 4

Separation during genetic fingerprinting

Answer 4

Fragments of DNA are separated according to size by gel electrophoresis

The gel is immersed in alkali to separate the double strands into single strands

Question 5

Hybridisation during genetic fingerprinting

Answer 5

DNA probes (radioactive/fluorescent) bind with VNTR

The probes have complementary base sequences to the VNTRs

The process uses different probes to bind with different DNA sequences

Question 6

Development of genetic fingerprinting

Answer 6

An x-ray film is out over the nylon membrane

The film is exposed to the radiation from the radioactive probes, or from fluorescent probes by which light is visually identified

These point correspond to the position of the DNA fragments as separated during electrophoresis, so a series of bars is revealed.

Question 7

Methods of producing DNA fragments

Answer 7

• Using Reverse Transcriptase
• Using Restriction Endonucleases
• Using the Gene Machine

Question 8

Using Reverse Transcriptase to produce DNA fragments

Answer 8

1. Cell that readily produces lots of a protein is selected due to a high amount of mRNA
2. mRNA acts as a template which reverse transcriptase acts upon, forming cDNA
3. cDNA is isolated by hydrolysis
4. Double stranded DNA is formed on the cDNA template by DNA polymerase. The double stranded DNA is the gene

Question 9

Using Restriction Endonucleases to produce DNA fragments

Answer 9

1. Restriction Endonucleases cut the DNA strand at a specific sequence known as a recognition site.
2. Can cut between two opposite base pairs producing blunt ends, or in the middle of the site producing sticky ends

Question 10

Using the ‘Gene Machine’ to produce DNA fragments

Answer 10

1. Desired amino acid sequence of the protein is determined, the mRNA codons are looked up and the complementary DNA triplets are worked out.
2. The desired nucleotide sequence is fed into a computer where it is checked for biosafety concerns.
3. The computer designs small, overlapping single strands of nucleotides, called oligonucleotides that can be assembled into the desired gene
4. In an automated process, each of the oligonucleotides are assembled one at a time in the required sequence.
5. The oligonucleotides are then joined together to make a gene. The gene has no introns and is replicated by the polymerase chain reaction.
6. The polymerase chain reaction constructs the complementary strand of nucleotides to make the required double stranded gene. Using sticky ends, the gene is inserted into a bacterial plasmid allowing it to be cloned and errors are rejected.

Advantages: Quick, accurate and no introns.

Question 11

Three Stages of the Polymerase Chain Reaction

Answer 11

1. Separation (95 Degrees)
2. Addition of primers (55 Degrees)
3. Synthesis of dna (72 Degrees)

Question 12

Components needed for the polymerase chain reaction

Answer 12

• DNA Nucleotides - to be added to form the cloned fragment
• DNA fragment(s) to be cloned
• Primers - to begin the addition of nucleotides, have base sequences complementary to the start and end of the fragment
• DNA polymerase - to add nucleotides
• Thermocycler - hold the mixture and change the heat accordingly

Question 13

First stage of the Polymerase chain reaction (95)

Answer 13

The thermocycler is heated to 95 degrees to break the hydrogen bonds between the DNA fragment, exposing the nucleotides

Question 14

Second stage of the Polymerase chain reaction (55)

Answer 14

The thermocycler is heated to 55 degrees because it is the optimum temperature for primers to be added to either end of the DNA fragment. The primers allow the addition of nucleotides to begin

Question 15

Third stage of the Polymerase chain reaction (72)

Answer 15

The thermocycler is heated to 72 degrees because it is the optimum temperature for DNA polymerase to act on the separated strand, joining nucleotides together and forming two new DNA fragments

Question 16

Advantages of In Vitro gene cloning (PCR)

Answer 16

• Extremely rapid, within a matter of hours 100 billion copies of a gene can be cloned through the polymerase chain reaction. In Vivo cloning would take weeks to produce the same
• Does not require living cells. All that is required is a base sequence - no complex time and effort or culturing techniques

Question 17

Advantages of In Vivo gene cloning (Plasmids)

Answer 17

• Almost no risk of contamination because they are cut by the same restriction endonulease and a contaminant will not be taken up by a plasmid. In Vitro requires a pure sample
• Very accurate. Few errors for the copied DNA. 20% of in vitro DNA is inaccurately copied.