Chapter 13 - DNA Manipulation Techniques

Question 1

Restriction enzyme (or restriction endonuclease)

Answer 1

A bacterial enzyme that recognises a short sequence of bases in a DNA molecule and cuts the DNA into fragments at specific restriction sites.

Question 2

Restriction site

Answer 2

A particular order of nucleotides

Question 3

DNA ligase

Answer 3

An enzyme that sticks DNA fragments together

Question 4

Blunt ends

Answer 4

Occur when restriction enzymes cut the two strands of a DNA molecule at points directly opposite each other.
These ends are not single stranded.

Question 5

Sticky ends

Answer 5

Occur when restriction enzymes cut one strand at one point, but the second at a point that is not directly opposite.
Produces single stranded ends, are complimentary.

Question 6

How are restriction enzymes named?

Answer 6

1st part: 3 letters derived from the scientific name of the microbe in which the restriction enzyme occurs. 1st letter = genus, 2nd and 3rd letters = specific name.

2nd part: Roman numberal (I, II, III) given to each different restriction enzyme from the same microbe.

Question 7

Define: Gene editing/genome editing/DNA editing

Answer 7

A process by which changes to the DNA sequence of genes can be achieved.

Question 8

What is CRISPR-Cas9 technology

Answer 8

Efficient, easy-to-use, inexpensive tool for precise gene editing to the genome of living cells.

Add, edit, modify, disable or delete DNA from the genome.

Question 9

How does CRISPR-Cas9 technology work?

Answer 9

Requires two tools: molecular ‘scissors’ and ‘guide’.
Scissors = Cas9 nuclease = can unwind double-stranded DNA and cut both strands at a precise location.
Guide = a segment of an artificially synthesised single strand of RNA (20 base sequence that is complimentary to part of the target DNA

Question 10

Two kinds of gene editing that can occur to repair the breaks form cutting of DNA?

Answer 10

1. Gene ‘knock in’ = a specifically designed DNA sequence is inserted into a precise location of the genome. Aims to fix faulty genes and restore their normal function.
2. Gene ‘kick out’ = random insertion or deletion of a segment, producing a frame shift mutation, either disabling a gene or can produce a STOP signal. Aims to disable or silence a gene.

Question 11

Ethical considerations for CRISPR-Cas9 technology

Answer 11

Possibilities of:
Incomplete editing
Inaccurate editing 
Errors
Modified organisms being affected indefinitely and passing genes to future generations, potentially affecting them in unexpected ways
Human embryos

Question 12

Gel electrophoresis

What and process?

Answer 12

Is a technique use to separate segments of DNA (on the basis of different lengths) along a gel, driven by a voltage drop.
Process:
1. The gel is composed of agarose which has a chemical, ethidium bromide (a dye that binds to DNA molecules) added to it. The gel has tiny little pores in it.
2. The gel is added to a mould which has wells at one end. DNA size standards are added to one well, and the DNA sample to be tested is added to another well.
3. The gel is placed in an electrophoresis bath, where it is covered in buffer (saltwater) solution.
4. DNA is negatively charged, so it moves toward the positive pole, when placed in an electric field.
5. The gel is viewed under ultraviolet (UV) light. Ehtidium bromide binds to the DNA, which makes it fluorescent pink under UV illumination.

Question 13

Southern blot technique -

Used for, brief steps?

Answer 13

Used to pick out one particular set of DNA fragments from a large sample of DNA fragments that have been sorted by electrophoresis.

Process:

1. DNA fragments transferred from gel to nitrocellulose filter by blotting.
2. Gel treated with denaturing agent for single stranded DNA.
3. Gel transferred to salt solution, nitrocellulose filter placed over gel.
4. Layers of absorbent paper placed over filter. Solution passes through gel and filter to paper, producing nitrocellulose filter with DNA fragments positioned exactly as on the gel.
5. Filter exposed to labelled probe for a specific gene. Probe will hybridise with a specific short sequence present on the gene.
6. DNA of interest is located by X-ray film, radioactive probe or fluorescence detection. Identified by a band on film.

Question 14

Making specific DNA fragments - two methods

Answer 14

1. A DNA synthesiser can be used to join nucleotide sub units in a predefined Ofer tho produce DNA segments. Products can be used a primer or probe.
2. Isolate mRNA from specific cells. Reverse transcriptase uses the mRNA as a template to build complimentary DNA (cDNA)

Question 15

Define DNA probe

Answer 15

A purified single stranded DNA fragment (base squeeze complimentary to target strand of DNA) that carries a radioactive isotope or a fluorescent dye to protect specific segments of DNA.

Question 16

Define hybridisation

Answer 16

Pairing between single-stranded complimentary DNA segments from organisms of the same or even different species.

Question 17

Reverse transcriptase

Answer 17

Makes DNA from an RNA template

Question 18

Polymerase chain reaction (PCR) - DNA amplification

Used for?
Special DNA polymerase used?
Process?

Typically 30-40 cycles used to amplify a DNA sample

Answer 18

PCR is used to clone (copy) a specific sequence of DNA without the need of a living cell.
Taq polymerase - can tolerate high temperatures for long periods of time.

Process:
1 DNA sample, DNA polymerase as single-stranded DNA primers are placed together in a 0.5mL plastic tube in a DNA thermocycler (a hearing block that can change temperature quickly)
2. Denature: heat sample to about 95dc. The strands of the DNA sample separate to make two single-stranded templates.
3. Bind primers: cool to 50-60dc. Primers bins to their complimentary DNA sequence.
4. Extend primers: heat to 72dc. The DNA polymerase start from the primer, moves along the DNA template and begins to add nucleotides so that two complete double strands are formed.