6.3.6: Genetic engineering

Question 1

Why is genetic engineering also known as recombinant DNA technology?

Answer 1

Because it involves combining DNA from different organisms.

Question 2

Briefly outline in four steps the process of genetic engineering.

Answer 2

1. The required gene is obtained.
2. A copy of the gene is placed inside a vector.
3. The vector carries the gene into a recipient cell.
4. The recipient expresses the novel gene.

Question 3

There are four ways that you could obtain the required gene. Describe the approach involving mRNA

Answer 3

• mRNA can be obtained from cells where the gene is being expressed.
• An enzyme, reverse transcriptase, can then catalyse the formation of a single strand of complementary DNA (cDNA) using the mRNA as a template.
• The addition of primers and DNA polymerase can make this cDNA into a double-stranded length of DNA, whose base sequence codes for the original protein.

Question 4

There are four ways that you could obtain the required gene. Describe the approach involving an automated polynucleotide synthesiser.

Answer 4

-If scientists know the nucleotide sequence of the gene, then the gene can be synthesised using an automated polynucleotide synthesiser.

Question 5

There are four ways that you could obtain the required gene. Describe the approach involving PCR.

Answer 5

-If scientists know the sequence of the gene, they can design PCR primers to amplify the gene from the genomic DNA.

Question 6

There are four ways that you could obtain the required gene. Describe the approach involving a DNA probe.

Answer 6

-A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes.

Question 7

Outline the process of using plasmids as vectors for the gene.

Answer 7

• Plasmids can be obtained from organisms such as bacteria and mixed with restriction enzymes that will cut the plasmid at specific recognition sites.
• The cut plasmid has exposed unpaired nucleotide bases, called sticky ends.
• If free nucleotide bases, complementary to the sticky ends of the plasmids, are added to the ends of the gene to be inserted, then the gene and cut plasmid should anneal (bind).
• DNA ligase enzyme catalyses the annealing

Question 8

How can a virus be used as a vector for the gene?

Answer 8

A gene may be sealed into an attenuated (weakened) virus that could carry it to a host cell.

Question 9

DNA does not easily cross the recipient cell’s membrane. Various methods can be used to aid the process. Describe how heat shock treatment can be used.

Answer 9

• If bacteria are subjected to alternating periods of cold (0 degrees celsius) and heat (42) in the presence of calcium chloride, their walls and membranes will become more porous and allow in the recombinant vector.
• This is because the positive calcium ions surround the negatively charged parts of both the DNA molecules and phospholipids in the cell membrane, thus reducing repulsion between the foreign DNA and the host cell membrane.

Question 10

DNA does not easily cross the recipient cell’s membrane. Various methods can be used to aid the process. Describe how electroporation can be used.

Answer 10

A high voltage pulse is applied to the cell to disrupt the membrane.

Question 11

DNA does not easily cross the recipient cell’s membrane. Various methods can be used to aid the process. Describe how electrofusion can be used.

Answer 11

Electrical fields help to introduce DNA into cells.

Question 12

DNA does not easily cross the recipient cell’s membrane. Various methods can be used to aid the process. Describe how transfection can be used.

Answer 12

DNA can be packaged into a bacteriophage, which can then transfect the host cell.

Question 13

DNA does not easily cross the recipient cell’s membrane. Various methods can be used to aid the process. Describe how the bacterium Agrobacterium tumefaciens can be used.

Answer 13

T1 (recombinant) plasmids are inserted into the bacterium Agrobacterium tumefaciens, which infects some plants and naturally inserts its genome into the host cell genomes.

Question 14

When might a direct method of introducing a gene into a recipient be used and how is this done?

Answer 14

• If plants are not susceptible to A.tumefaciens, then direct methods can be used.
• Small pieces of gold or tungsten are coated with the DNA and shot into the plant cells.
• This is called a ‘gene gun’.

Question 15

Reverse transcriptase enzymes are useful in genetic engineering. Describe how retroviruses such as HIV use these enzymes.

Answer 15

• Retroviruses, such as HIV contain RNA that they inject into the host genome
• They have reverse transcriptase enzymes that catalyse the production of cRNA (complementary DNA) using their RNA as a template.
• This is the reverse of transcription.

Question 16

How do bacteria and Archaea protect themselves from phage viruses?

Answer 16

• Bacteria and Archaea have restriction enzymes, called restriction endonucleases, to protect them from attack by phage viruses.
• These enzymes cut up the foreign viral DNA, bu a process called restriction.
• This prevents the viruses from making copies of themselves.

Question 17

How is prokaryotic DNA protected from the action of restriction endonucleases?

Answer 17

-By being methylated at the recognition sites.

Question 18

How are restriction enzymes useful to molecular biology and biotechnology?

Answer 18

• They are useful as molecular scissors as they recognise specific sequences within a length of DNA and cleave the molecule there.
• Some leave sticky ends by making a staggered cut.
• Others make a cut that produces blunt ends.

Question 19

Which types of sequences do restriction enzymes always recognise?

Answer 19

A palindromic sequence.

Question 20

What do some restriction enzymes need as cofactors?

Answer 20

magnesium ions.

Question 21

What are ligase enzymes used for in molecular biology?

Answer 21

• To join DNA fragments.
• It catalyses condensation reactions that join the sugar groups and phosphate groups of the DNA backbone.
• These enzymes catalyse such reactions during DNA replication in cells and are also used in the PCR.

Question 22

Where is the gene for insulin production obtained from?

Answer 22

-Scientists can obtain mRNA from beta cells of islets of Langerhans in the human pancreas, where insulin is made.

Question 23

Outline the process of using genetically modified bacteria to produce insulin.
Step 1:

Answer 23

1. Adding reverse transcriptase enzymes makes a single strand of cDNA and treatment with DNA polymerase makes a double strand- the gene.

Question 24

Outline the process of using genetically modified bacteria to produce insulin.
Step 1: Adding reverse transcriptase enzymes makes a single strand of cDNA and treatment with DNA

Step 2:

Answer 24

2. Addition of free unpaired nucleotides at the ends of the DNA produces sticky ends.

Question 25

Outline the process of using genetically modified bacteria to produce insulin.
Step 2: Addition of free unpaired nucleotides at the ends of the DNA produces sticky ends.

Step 3:

Answer 25

3. Now, with the help of ligase enzymes, the insulin gene can be inserted into the plasmids extracted from E.coli bacteria. These are now called recombinant plasmids, as they now contain inserted DNA.

Question 26

Outline the process of using genetically modified bacteria to produce insulin.
Step 3: Now, with the help of ligase enzymes, the insulin gene can be inserted into the plasmids extracted from E.coli bacteria. These are now called recombinant plasmids, as they now contain inserted DNA.

Step 4:

Answer 26

4. E.coli bacteria are mixed with recombinant plasmids and subjected to heat shock in the presence of calcium chloride ions so that they will take up the plasmids.

Question 27

What safety precaution is taken to prevent transformed (transgenic) bacteria that have resistance to some antibiotics from ‘escaping’ into the wild?

Answer 27

• They also have a gene knocked out, which means they cannot synthesise a particular nutrient.
• They survive in the laboratory where they are given that nutrient in their growth medium, but will not survive outside of the laboratory.