Recombinant DNA technology

Question 1

1. What is recombinant DNA technology?

Answer 1

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

Question 2

2. What property of DNA allows recombinant DNA technology to happen? Explain why.

Answer 2

The genetic code is universal, as are the transcription and translation mechanisms, so the transferred DNA can be transcribed into mRNA, then translated into polypeptides within cells of the recipient organism. This organism is now transgenic.

Question 3

3. What is the role of reverse transcriptase in
   recombinant DNA technology?

Answer 3

It produces cDNA using mRNA.

Question 4

4. How is a DNA double helix produced from cDNA.

Answer 4

DNA polymerase joins adjacent DNA nucleotides in a phosphodiester bond to produce the complimentary strand of DNA.

Question 5

5. What is the role of restriction endonucleases in making DNA fragments?

Answer 5

The enzymes used to cut a fragment of DNA containing the desired gene from the organisms DNA.

Question 6

6. Why is it an advantage to produce DNA fragments
   using mRNA and reverse transcriptase, than cutting a gene out using restriction enzymes?

Answer 6

mRNA does not contain introns, so the cDNA produced will only contain exons. Some organisms (prokaryotes) can’t splice introns out of mRNA; There is much more mRNA in a cell than DNA for a particular protein; mRNA will be found in the cell that specifically makes the protein;

Question 7

7. What is a gene machine?

Answer 7

An automated way of producing DNA fragments with the correct DNA base sequence.

Question 8

8. What is the benefit of using the gene machine to
   produce a DNA fragment, rather than using reserve transcriptase?

Answer 8

There are more steps involved in isolating mRNA; Faster to use gene machine than all the enzyme-catalysed reactions involving reverse transcriptase;

Question 9

1. What is amplification of DNA?

Answer 9

Making more copies of a fragment of DNA.

Question 10

2. What two techniques are there of amplifying DNA?

Answer 10

In vitro amplification – (outside organisms) using the Polymerase Chain Reaction (PCR). In vivo amplification – (inside organisms) inserting the fragment into the genome of a host cell and culturing the host cell to produce more cells and therefore more copies of the gene.

Question 11

3. Describe the polymerase chain reaction.

Answer 11

Heat DNA to 90oC; Breaks hydrogen bonds and separates strands; Add primers; Add nucleotides; Cool; to allow binding of primers to nucleotides; increase temperature to the optimum for DNA polymerase; DNA polymerase joins adjacent DNA nucleotides together into a new strand; Repeat cycle many times;

Question 12

4. Why does the DNA replication in the PCR eventually stop?

Answer 12

There are a limited number of primers and nucleotides available and these run out; so there is nothing to start/make the complimentary chains

Question 13

5. What is the role of primers in the PCR?

Answer 13

Short sequences of nucleotides with complementary bases to the end of the DNA fragments being copied by PCR. They bind to the start of the sequence to be copied and allow the DNA polymerase to join nucleotides to produce the complimentary strand.

Question 14

6. Why do primers only bind to specific sequences?

Answer 14

Each primer have a specific base sequence; That is complementary to the start of the gene to be copied;

Question 15

7. What are the advantages of PCR over in vivo amplification?

Answer 15

PCR can rapidly produce many copies. PCR does not require the use of living cells.

Question 16

1. During in vivo amplification, describe how a gene could be inserted into a plasmid.

Answer 16

Cut the plasmid using the same restriction endonuclease as the fragment; So that both have complementary sticky ends; Mix together the fragments and cut plasmids and add the enzyme DNA ligase to join the complementary sticky ends;

Question 17

2. Explain why it is important to use the same restriction enzyme on both pieces of DNA.

Answer 17

To cut the DNA at the same recognition sequence; so the sticky ends are complementary;

Question 18

3. Explain how sticky ends are useful in genetic engineering.

Answer 18

Joining two pieces of DNA; By complementary base-pairing;

Question 19

4. Why do DNA fragments need a promoter and terminator regions adding to them?

Answer 19

A fragment created using reverse transcriptase and mRNA has no promoter and terminator regions. The promoter and terminator regions tell RNA polymerase where to start and finish transcription, so they need to be incorporated into the fragment that is being inserted into an organism to produce a specific protein.

Question 20

. What is the role of a vector?

Answer 20

Vectors are used to transport the DNA fragment into the host cell.

Question 21

6. What are the roles of enzymes in the insertion
   of a DNA fragment into plasmid/vector?

Answer 21

Restriction enzymes cut the plasmid/vector; DNA ligase joins the sticky ends of the DNA fragment to the plasmid/vector via phosphodiester bonds.

Question 22

7. Once a fragment of DNA has been inserted into a plasmid/vector. How is it amplified?

Answer 22

The plasmid/vector is taken up by the host cell; the host perform cell division producing many copies of the desired fragment;

Question 23

8. What is the transformation of a host cell?

Answer 23

The uptake of a new DNA fragment from a different organism/species, using a vector to insert the fragment into the organisms genome. The host cell should now be able to produce a polypeptide inserted fragment codes for

Question 24

9. What is the role of marker genes?

Answer 24

Marker genes are used to detect whether the DNA fragment has been successfully inserted in to the hosts cells/to identify recombinant cells.

Question 25

10. What is a genetically modified (GM) organism?

Answer 25

An organism that has had a DNA fragment from another organism/species inserted into the genome, so that it can now produce the polypeptide the DNA fragment codes for.

Question 26

11. Give examples of how marker genes can identify genetically modified (GM) organisms.

Answer 26

Use a plasmid with two antibiotic resistance genes; add the target gene into the middle of the first marker gene. Grow the bacteria on agar containing the antibiotic which the second marker gene gives resistance to. Create a replica plate on agar containing the antibiotic that the first resistance gene gives resistance to. Any colonies present on the first plate that did not grow on the replica plate must be recombinant. Alternatively use a different marker gene for the second marker e.g. green fluorescent protein or an enzyme that makes a coloured product. No replica plate is needed for these as the colonies will be a different colour/fluoresce under UV ligh

Question 27

12. Explain why it is important that vectors are
    added to the embryos of multicellular organism (plants/animals)

Answer 27

If injected into embryo, gene gets into most of cells of organism; So gets into cells that need to make the protein; As the DNA will be replicated with the host DNA inside the nucleus; Then passed to new cells during mitosis; to produce genetically identical cells;

Question 28

13. Explain the role of recombinant DNA technology in gene therapy.

Answer 28

DNA fragments containing the normal form of a gene can be added to a virus; the virus can be used to infect an individual that has a faulty version of the gene; the virus inserts the normal form of the gene into the individuals genome of target cells; the target cells can now make the normal protein for a limited period;