Identify target sequences, match restriction-enzyme cutting with ligase joining, trace recombinant constructs into vectors or hosts, and link restriction sites to gel-fragment lengths.
Gel electrophoresis is used to separate DNA fragments of different lengths.
The rate at which the DNA fragments move through the gel depends on several factors including:
- molecular size of the DNA fragment
- shape of the DNA fragment
- concentration of the gel.
7(a)(i)
C — restriction enzyme.
A is incorrect because DNA polymerase is used to synthesise DNA. B is incorrect because integrase inserts DNA into other DNA. D is incorrect because reverse transcriptase synthesises DNA using an RNA template. (1)
7(a)(ii)
Any three of:
- restriction enzymes recognise specific base sequences;
(1)
- recognition sites are not equally spaced along the DNA;
(1)
- the enzyme hydrolyses/breaks phosphodiester bonds;
(1)
- therefore different-sized fragments move different distances/speeds through the gel. (1)
Accept different enzymes cut at different sites / different cutting sites result in different length fragments.
7(b)(i)
- 0.2. (1)
- 794 / 871 / 873 / 1000. (1)
7(b)(ii)
Indication of spot positioned between the origin and the 10 000 spot. Do not accept if clearly overlapping. Ecf from 7(b)(i): if answer in 7(b)(i) is greater than 0.25, the spot would be below the 10 000 spot. (1)
7(c)
More molecules/bonds, smaller spaces or fewer spaces, so higher resistance/friction;
accept harder to move for higher resistance. (1)
7(d)(i)
Any two of:
- bacterial genome/chromosome / bacterial nucleoid;
(1)
- plasmid;
(1)
- mitochondrial DNA / mtDNA;
(1)
- chloroplast DNA / cpDNA;
(1)
Accept cccDNA formed by some viruses inside cell nuclei, or viral extrachromosomal circular DNA. Do not accept ribosomes, cytoplasm, or viruses alone.
7(d)(ii)
Any two of:
- linear DNA has 3′ and 5′ ends but circular DNA does not;
(1)
- linear DNA is associated with more proteins/histones than circular DNA;
accept converse / circular DNA is supercoiled but linear DNA is not;
(1)
- linear DNA will have unbound phosphate/deoxyribose but circular DNA will not;
(1)
- linear DNA will have one fewer phosphodiester bond than circular DNA with the same number of mononucleotides. (1)
Which enzyme is used to cut the DNA into fragments?
DNA polymerase
integrase
restriction enzyme
reverse transcriptase
C
Explain why the use of an enzyme to cut the DNA results in fragments, of different lengths, that can be separated by gel electrophoresis.
An explanation that includes three the following points:
- because restriction enzymes recognise specific (base) sequences (1)
- because these recognition sites are not equally spaced along the DNA (1)
- the enzyme hydrolyses the phosphodiester bonds (1)
- therefore different sized fragments move different distances through the gel (1)
IGNORE incorrectly named enzyme from part (i)
ACCEPT different enzymes cut at different sites
ACCEPT different cutting sites will result in different length fragments
ACCEPT breaks
ACCEPT different speeds
(3)