21.2 In Vivo gene cloning - use of vectors

Question 1

What are the two methods of cloning DNA fragments

Answer 1

1. In Vivo - Using vectors to transfer them into host cells

2. Using the polymerase chain reaction

Question 2

What are the sequences cut by restriction endonucleases called

Answer 2

Recognition sites

Question 3

What happens if the recognition site is cut in a staggered fashion

Answer 3

It produces sticky ends, which means the cut ends of the DNA double strand are left with a single strand which is a few nucleotides long.

Question 4

What enzyme joins the phosphate-sugar framework of the two sections of DNA as one

Answer 4

DNA ligase

Question 5

What is involved in the preparation of the DNA fragment for insertion

Answer 5

It involves the addition of extra lengths of DNA.

For the transcription of any gene to take place, RNA polymerase must attach to the DNA near a gene. The binding site is a region of DNA, known as a promoter.

Equally, a region that releases the RNA polymerase is added - known as a terminator region.

Question 6

What is a vector

Answer 6

A carrying unit of the DNA fragment into another cell

Question 7

What is the most commonly used vector

Answer 7

A plasmid

Question 8

What is a plasmid

Answer 8

A small circular length of DNA found in bacteria that is separate from the main bacterial DNA.

Question 9

What to plasmids usually contain

Answer 9

Genes for antibiotic resistance

Question 10

What does the process of insertion into a vector involve

Answer 10

Once an appropriate fragment of DNa has been cut from the rest, and the promoter and terminator regions added, the next step is to join it into a vector.

Restriction endonucleases are used which open up the plasmid and DNA ligase joins the DNA fragment to the plasmid. These now contain recombinant DNA

Question 11

What does the introduction of DNA into host cells involve

Answer 11

Once the DNA has been incorporated into at least some plasmids, they’re reintroduced back into bacterial cells. This is called transformation

Question 12

What conditions are required for introduction to occur

Answer 12

The plasmids and bacterial cells are mixed together in a medium containing calcium ions. Temperature changes also occur. This makes the membrane permeable, allowing the plasmids to pass through into the cytoplasm.

Question 13

Why do only some of the bacterial cells possess the DNA fragments with the desired gene for the desired protein

Answer 13

1. Only a few bacterial cells take up the plasmids when they are mixed together
2. Some plasmids will have closed up again without incorporating the DNA fragment
3. Sometimes the DNA fragments join together to form its own plasmid

Question 14

What are the different marker genes1

Answer 14

1. Antibiotic resistance marker gene
2. Fluorscent marker gene
3. Enzyme markers