3.8 - 3.8.4 - 3.8.4.1 Recombinant DNA technology (A-level only)

Question 1

What does recombinant DNA technology involve?

Answer 1

The transfer of fragments of DNA from one organism, or species, to another.

Question 2

Since the genetic code is _ _ _ _ _ _ _ _ _, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism to produce a protein in the cells of the recipient organism.

Answer 2

Universal (the same DNA based triplets code for the same amino acids in all living things).

Question 3

The recipient and donor organisms don’t have to be from the same species. True or false for transferring fragments of DNA from one organism, or species, to another?

Answer 3

True and this can be very useful.

Question 4

Organisms that contain transferred DNA are known as what?

Answer 4

Transgenic organisms.

Question 5

Fragments of DNA can be produced in three different ways, including:

Answer 5

• Conversion of mRNA to cDNA, using reverse transcriptase.
• Using restriction enzymes to cut a fragment containing the desired gene from DNA.
• Creating the gene in a ‘gene machine’.

Question 6

If you use reverse transcriptase to produce DNA fragments,
Why is it difficult to obtain a DNA fragment containing the target gene but easier to contain many mRNA molecules which are complementary to the gene?

Answer 6

It is difficult to obtain a DNA fragment containing the target gene as most cells only contain two copies of each gene.
mRNA is often easier to obtain as they can contain many mRNA molecules which are complementary to the gene.

Question 7

If you use reverse transcriptase to produce DNA fragments,
What are the mRNA molecules used as? to make what?

Answer 7

Templates to make lots of DNA.

Question 8

If you use reverse transcriptase to produce DNA fragments,
What does the enzyme, reverse transcriptase make DNA from?

Answer 8

An RNA template.

Question 9

If you use reverse transcriptase to produce DNA fragments,
What is the DNA produced called?

Answer 9

Complementary DNA (cDNA).

Question 10

If you use reverse transcriptase to produce DNA fragments,
What is the process of getting the cDNA?

Answer 10

mRNA is first isolated from cells, then mixed with free DNA nucleotides and reverse transcriptase, the reverse transcriptase uses the mRNA as a template to synthesise a new strand of cDNA.

Question 11

If you use restriction endonuclease enzymes to produce DNA fragments,
What are palindromic sequences of nucleotides?

Answer 11

Sequences which consist of antiparallel base pairs (base pairs that read the same in opposite directions).

Question 12

If you use reverse transcriptase to produce DNA fragments,
What are restriction endonuclease?

Answer 12

Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places.

Question 13

If you use reverse transcriptase to produce DNA fragments,
What do different restriction endonuclease cut at?

Answer 13

Different specific recognition sequences, because the shape of the recognition sequence is complementary to the enzymes active site.

Question 14

If you use reverse transcriptase to produce DNA fragments,
If recognition sequences are present at either side of the DNA fragment you want, what can you use restriction endonucleases to separate?

Answer 14

It from the rest of the DNA.

Question 15

If you use reverse transcriptase to produce DNA fragments,
When the DNA sample is incubated with the specific restriction endonuclease, which cuts the DNA fragment out via a hydrolysis reaction, sometimes, the cut leaves what?

Answer 15

The cut leaves sticky ends.

Question 16

If you use reverse transcriptase to produce DNA fragments,
What are sticky ends?

Answer 16

Small tails of unpaired bases at each end of the fragment.

Question 17

If you use reverse transcriptase to produce DNA fragments,
What can sticky ends be used to bind (anneal)?

Answer 17

The DNA fragment to another piece of DNA that has sticky ends with complementary sequences.

Question 18

If you use a ‘Gene Machine’ to produce DNA fragments,
There is no need for a pre-existing DNA template, why is this?

Answer 18

Because technology has been developed so that fragments of DNA can be synthesised from scratch. A database contains the necessary information to produce the DNA fragment. This means that the DNA sequence does not have to exist naturally - any sequence can be made.

Question 19

If you use reverse transcriptase to produce DNA fragments,
How is it done?

Answer 19

• The sequence that is required is designed (if one does not already exist).
• The first nucleotide in the sequence is fixed to some sort of support, e.g. a bead.
• Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups. Protecting groups make sure that the nucleotides are joined at the right points, to prevent unwanted branching.
• Short sections of DNA called oligonucleotides, roughly 20 nucleotides long, are produced. Once these are complete, they are broken off from the support and all the protecting groups are removed. The oligonucleotides can then be joined together to make longer DNA fragments.

Question 22

Fragments of DNA can be amplified by in vitro and in vivo techniques.
The principles of the polymerase chain reaction (PCR) as an in vitro method to amplify DNA fragments.
The culture of transformed host cells as an in vivo method to amplify DNA fragments.
- The addition of promoter and terminator regions to the fragments of DNA.
- The use of restriction endonucleases and ligases to insert fragments of DNA into vectors. Transformation of host cells using these vectors.
- The use of marker genes to detect genetically modified (GM) cells or organisms.