Manipulation of Genes and Cells

Question 1

What enzyme is used to isolate and purify a gene from a large DNA molecule?

Answer 1

Restriction nucleases

Question 2

What reaction does a nuclease catalyse?

Answer 2

Hydrolysis of a phosphodiester bond in a nucleic acid.

Question 3

What is a special property of restriction nucleases?

Answer 3

They cut the double-stranded DNA only at particular sites.

Question 4

What is the importance of these restriction nucleases?

Answer 4

They can be used to produce a reproducible set of specific DNA fragments from any genome

Question 5

Where are these nucleases obtained from?

Answer 5

They are obtained from bacteria.

Question 6

How can DNA fragments be separated from one another?

Answer 6

Gel electrophoresis

Question 7

How are DNA fragments separated in electrophoresis?

Answer 7

By their length

Question 8

How does electrophoresis work?

Answer 8

A voltage is applied across the gel. The DNA molecule is negatively charged and the larger fragments will migrate more slowly because their progress is impeded.

Question 9

How is DNA sequenced?

Answer 9

Dideoxy method

Question 10

What happens in the dideoxy method?

Answer 10

DNA polymerase is used to make partial copies of the DNA fragment to be sequenced. This replication reactions are performed under conditions to ensure that the new DNA strands terminate when a given nucleotide (A, G , C or T) are reached. This method produces a collection of different DNA copies that terminate at every position in the original DNA and differ in length by a single nucleotide. These can then be separated using electrophoresis and ordered.

Question 11

What is nucleic acid hybridisation?

Answer 11

A technique in which single-stranded nucleic acids (DNA or RNA) are allowed to interact so that complexes called hybrids are formed by molecules with similar or complementary sequences.

Question 12

Why is the high specificity of nucleic acid hybridisation important?

Answer 12

We can determine the exact nucleotide change e.g. in sickle cell anaemia. Makes scanning through the human genome a lot more simple.

Question 13

What is cloning of DNA?

Answer 13

It is the act of making identical copies of a DNA molecule.

Question 14

What is transformation?

Answer 14

Introduce DNA to be copied into rapidly dividing bacterium. For example E. coli can be coaxed to take up recombinant DNA made up in the laboratory.

Question 15

What happens when the modified DNA is inside the recipient cell?

Answer 15

The donor DNA can become a part of the recipient genome (e.g. through homologous recombination) or it can be maintained as a piece of DNA independent to that of the bacterial chromosome.

Question 16

Why are plasmids useful?

Answer 16

Because it is easier to manipulate, copy and purify recombinant DNA when it is maintained with certainty as an independent molecule., separate from the bacterial chromosome.

Question 17

What are useful properties of a plasmid?

Answer 17

• it has a cutting site for a convenient restriction nuclease so that the plasmid can be opened and a foreign DNA fragment can be inserted.
• They contain some selectable property such as antibiotic resistance which means that the bacteria that take up the recombinant DNA are easily identified.

Question 18

How can the DNA fragment be recovered from the plasmid DNA?

Answer 18

By being cleanly cut out using a restriction enzyme and then separated from plasmid DNA by gel electrophoresis.

Question 19

How are human genomic libraries constructed?

Answer 19

Using restriction nucleases and ligase.

Question 20

What compromises of a human genomic library?

Answer 20

A set of bacteria, each carrying a different small fragment of human DNA

Question 21

What is different about the cDNA library?

Answer 21

The complementary DNA library does not contain any introns.

Question 22

How is the cDNA library created?

Answer 22

total mRNA is extracted from cells and a DNA copy is made using reverse transcriptase. DNA polymerase is then used to make a complementary DNA strand. mRNA does not contain any intron information.

Question 23

What is PCR used for?

Answer 23

The amplification of selected DNA sequences to make multiple copies. Special heat resistant DNA polymerase is used so it will not denature as DNA strands are heated after each cycle.

Question 24

Describe DNA engineering

Answer 24

Splice together DNA fragments derived from different sources. After each DNA insertion step, recombinant plasmid is cloned to purify and amplify new DNA. Recombinant molecule is then cut once with a restriction nuclease and used as a cloning vector for next DNA fragment.

Question 25

What is the ultimate test for the function of a mutated gene?

Answer 25

Insert it into the genome of an organism and see the effect that it has. These are known as transgenic organisms, for example in mice.

Question 26

Name some different types of microscopy

Answer 26

• fluorescence microscopy
• confocal microscopy
• light microscope for cells