MCD - NAGE

Question 1

Outline cell based DNA cloning

Answer 1

The target DNA and the replicon (plasmid) with restriction endonucleases, making complimentary sticky ends. This cuts the DNA at palindromes. The DNA fragments are joined using DNA ligase. Recombinant DNA undergoes transformatioj into host cells and then is selectively propagated on an agar plate, using an antibiotic resistance marker in the plasmid. Only host cells that have taken up the replicon will survive. The recombinant DNA is isolated.

Question 2

Describe the function of restriction endonucleases

Answer 2

Type II cleave DNA at specific recognition sites called palindromes, 4-8 bases long. They are used in the bacterial immune system - bacterial DNA is protected as it is methylated. Blunt or sticky ends may be produced. The longer the recognition site, the less frequently it will be in DNA and the less the restriction endonuclease will be used.

Question 3

Describe the use of electrophoresis to separate DNA fragments

Answer 3

DNA is negatively charged due to phosphate groups - therefore the DNA molecules will move through the agar plate to the anode (+ve) when an electrical force is applied. Smaller fragments travel faster and further than larger fragments¬ as they are retarded less. DNA is then isolated from the gel or transferred to a membrane to form a replica for hybridisation.

Question 4

What is a hybridisation assay?

Answer 4

This is where target DNA is immoblised on a solid support that readily binds to a single stranded nucleic acid. These are then hybridised with a solution of radioactively or flourescently labeled probes. The sample is incubated and washed, then the DNA can be isolated. Used in blotting techniques.

Question 5

What does a southern blot hybridisation use?

Answer 5

A DNA target and a DNA probe

Question 6

What does a northern blot hybridisation use?

Answer 6

RNA target and a DNA probe

Question 7

What does a colony blot hybridisation use?

Answer 7

Bacterial DNA target and DNA probe

Question 8

What does tissue in situ hybridisation use?

Answer 8

A RNA target and RNA probe

Question 9

What does chromosome in situ hybridisation use?

Answer 9

A chromosome target and DNA probe

Question 10

Define hybridisation stringency

Answer 10

The power to distinguish between related sequences

Question 11

Describe how to denature probe DNA and what effects the energy required to denature the DNA.

Answer 11

Probe DNA is denatured by heating to provide energy to break hydrogen bonds holding the two strands together. The energy needed depends on strand length (longer strand means more energy), proportion of C and G (more C and G means more energy as these have 3 hydrogen bonds between the bases) and the presence of cations (cations stabilise the DNA complex so more energy is required, whereas denaturants such as urea destabilise the DNA).

Question 12

Define the ‘melting temperature’ (Tm) for DNA.

Answer 12

The midpoint temperature of transition from double stranded to single stranded forms of nucleic acid.

Question 13

What temperature¬ relative to Tm¬ is hybridisation carried out at?

Answer 13

Hybridisation is carried out at temperatures 25 degrees below Tm

Question 14

What affects hybridisation stringency and how?

Answer 14

Hybridisation stringency increases as the temperature increases, and increases as the Na+ concentration decreases. This is because the annealing of hybrids is dictated.

Question 15

What does low hybridisation stringency cause?

Answer 15

Annealing between strands of DNA that have some degree of mismatch between bases.

Question 16

What does high hybridisation stringency cause?

Answer 16

Duplexes form between the strands with perfect one to one complimentarity only.

Question 17

Describe the process of PCR

Answer 17

Stage 1: denaturing of DNA by heating to 94 degrees
Stage 2: annealing of primers by lowering the temperature to 50-60 degrees.
Stage 3: extension of the probe strand by Taq polymerase at 72 degrees. This works in the 5’ to 3’ direction. Taq polymerase is a hardy enzyme - not denatured by the temperature changes.

Question 18

Describe the three features a primer must have

Answer 18

• The length of a primer is 20 nuceleotides to ensure it is specific to a sequence
• The % of GC and length must give a equal Tm for each primer, and there must be no tandem repeats of nucleotides so hairpins aren’t formed.
• Complimentarity of the bases at the 3’ ends must be prevented, or primers will bind together to form primer dimers.

Question 19

List some of the applications of PCR

Answer 19

• Typing genetic markers (restriction fragment length polymorphisms)
• Detecting point mutations
• cDNA cloning
• Genome walking
• Introducing mutations experimentally
• DNA sequencing
• Microarrays

Question 20

How do DNA microarrays work?

Answer 20

Oglionucleotides, representing single genes, are robotically arrayed on a solid surface, and the microarray will fluoresce different colours depending on whether the gene is expressed in different organisms. mRNA is extracted from a cell and converted to cDNA using reverse transcriptase. cDNA is attatched fluorescent tags and washed over the sample. The cDNA hybridises with the immobilised oglionucleotides, and the expression of the genes can be determined by the colour and the intensity of the fluorescence.

Question 21

What are the applications of microarrays?

Answer 21

SNP detection arrays look for single nucleotide polymorphisms in the genome of populations. Expression profiling allows the expression of different genes to be studied, useful to identify the genes causing diseases and therefore target treatments towards those diseases.

Question 22

What is the difference between a codon and a triplet?

Answer 22

A codon is found in mRNA while a triplet is found in the DNA sequence.

Question 23

What are the dimentions of the double helix?

Answer 23

10 bases per helical turn, 2nm wide.

Major and minor groove present.

Question 24

What is a karyotype?

Answer 24

An organised profile of someones chromosomes

Question 25

What is the lowest level of packaging of DNA?

Answer 25

Nucleosomes - DNA wrapped around 8 histones in a left handed superhelix. Histones are positively charged, with 150 base pairs of DNA wrapped around the histones. Causes a 7 fold condensation.

Question 26

How are nucleosomes packed?

Answer 26

In 30nm fibers, giving a 40 fold condensation.

Question 27

Explain the process of semiconservative replication

Answer 27

• DNA helicase unzips the DNA helix using ATP.
• DNA primase synthesises RNA primers which bind to exposed DNA and act as a start point for DNA polymerase.
• DNA polymerase works in the 3’ to 5’ direction, so the 5’ to 3’ strand is the leading strand, and is synthesised in one swift motion. DNA polymerase is held on by a sliding clamp, and energy for the action of DNA polymerase is provided by the hydrolysis of dNTPs (deoxynucleotide triphosphates).
• At the lagging strand, many primers are needed and Okasaki fragments are made.
• Primers are removed by a ribonuclease using 5’ to 3’ exonuclease acrivity. Repair DNA polymerase replaces the RNA with DNA and DNA ligase joins the fragments together.

Question 28

What is the method of proof reading in DNA replication?

Answer 28

DNA polymerase checks the previous nucleotide before adding a new one, and if necessary removes it by its 3’ to 5’ exonuclease activity.

Question 29

How does replication occur in E.coli?

Answer 29

Begins from the origin point ori C, two replication forks meet at the other side of the chromosome. This is bidirectional replication.

Question 30

How does replication occur in the eukaryotic genome?

Answer 30

There are multiple replication origins, and each origin gives bidirectional replication forks. Replication finishes when the forks meet.

Question 31

Summarise the cell cycle

Answer 31

Gap phase 1, synthesis phase, gap phase 2, and mitosis (prophase, metaphase, anaphase, telophase)