Session 12: Introduction to Molecular Techniques

Question 1

Name three techniques that facilitate the analysis of human DNA

Answer 1

Restriction endonucleases
Cloning of DNA
DNA probes

Question 2

What are restriction endonucleases?

Answer 2

Enzymes that are able to cut huge ds DNA molecules into defined fragments

Question 3

What does cloning of DNA enable us to do?

Answer 3

Amplify specific nucleotide sequences

Question 4

What do DNA probes enable us to do?

Answer 4

Identify and manipulate the nucleotide sequence of interest

Question 5

Why do bacteria produce endonucleases?

Answer 5

To protect their own DNA by recognising and degrading foreign DNA

Question 6

Restriction enzymes cleave dsDNA so as to produce __________ on one end and a _________ on the other

Answer 6

3’ OH group

5’ Phosphate group

Question 7

TaqI is a restriction enzyme that forms staggered cuts that produce what?

Answer 7

“Sticky” ends

Question 8

What are “sticky” ends that are produced by restriction endonucleases?

Answer 8

The resulting fragments of single stranded DNA produced after cleavage are complementary to each other

Question 9

HaeIII produces fragments that have “blunt” ends, what does this mean?

Answer 9

The fragments produce ends that are double-stranded and therefore do not form hydrogen bonds with each other

Question 10

What does DNA ligase do?

Answer 10

Covalently joins “sticky” ends of DNA fragments together

Question 11

What is DNA gel electrophoresis?

Answer 11

A technique that allows us to separate DNA fragments based on their size or shape

Question 12

How does DNA gel electrophoresis work?

Answer 12

Samples are loaded into a buffered gel and more towards the positively charged electrode due to their negative charge
The smaller fragments travel further than the larger fragments

Question 13

DNA is ________ charged and will move towards the _________ if placed in an electric field

Answer 13

Negatively

Anode (+)

Question 14

What 4 things are required for gel electrophoresis?

Answer 14

1) Gel
2) Buffer
3) Power supply
4) Stain/detection

Question 15

What is the purpose of the buffer used in DNA gel electrophoresis?

Answer 15

To keep the pH constant and allow charge on the DNA samples across the gel

Question 16

Give four reasons why we may use restriction analysis

Answer 16

To investigate the size of DNA fragments
To investigate mutations
To investigate DNA variation e.g. DNA fingerprinting
To clone DNA

Question 17

What are the four basic steps of gene cloning?

Answer 17

Isolate the relevant gene of interest
Insert gene of interest into a plasmid vector
Introduce recombinant DNA molecule into suitable host cells
Identify and isolate the clone containing DNA of interest

Question 18

Why might DNA cloning be important?

Answer 18

To clone useful proteins such as insulin
To find out what genes do
Genetic screening
Gene therapy (Potentially?)

Question 19

When cloning eukaryotic genes would we overcome the fact that eukaryotic DNA contains both introns and exons?

Answer 19

Clone using the mRNA and reverse transcribe it to produce cDNA before placing into plasmid vector

Question 20

What is Polymerase Chain Reaction (PCR)?

Answer 20

Process by which target DNA is amplified exponentially using forward and reverse primers to define the region to be copied

Question 21

What might PCR be used for?

Answer 21

To amplify specific regions of a DNA fragment
To investigate single base pair mutations
To investigate small deletions or insertions
To investigate variation, genetic relationships

Question 22

What are the three cyclical steps of PCR and their temperatures?

Answer 22

1) Denature at 95oC
2) Anneal at 55oC
3) Polymerise at 75oC

Question 23

What are the types of DNA hybridisation?

Answer 23

Southern blotting (DNA)
Northern blotting (RNA)

Question 24

Why would we use Southern Hybridisation?

Answer 24

Looking at:

• gene structure (deletions, duplications) (CF)
• gene expansions, triplet repeats (Huntington’s)
• mutations in genetic tests (Sickle cell)
• Variation (Genetic fingerprinting)

Question 25

Do the DNA probes used in blotting have to have 100% similarity to the target sequence?

Answer 25

No, they do not have to have 100% complimentarity to the sequence

Question 26

Do probes have to completely align with the target sequence?

Answer 26

No, they don’t have to align, they can overlap and still detect the target sequence

Question 27

Do probes affect the position of the target sequence as it appears as banding on the gel?

Answer 27

No, no affect on position of banding we detect on the gel

Question 28

When we want to separate a protein based on their size, why do we first need to denature the protein in SDS?

Answer 28

To break the hydrogen bonds so that we have the protein as a linear polypeptide chain and they have a similar charge to mass ratio so are only separated based on their size and not its charge or shape

Question 29

When are 2D blots particularly useful?

Answer 29

When analysing the whole cell or whole tissue

Question 30

What are the stages in involved in Southern blotting?

Answer 30

• DNA gel electrophoresis
• Transfer of fragments to a membrane
• Hybridisation of a probe to detect specific piece of DNA
• Visualisation of labelled probe

Question 31

Explain how Sanger Chain Termination works

Answer 31

dideoxyNTPs can be used along with labelled primers, initiate DNA synthesis and analyse the encorporation of the ddNTPs in the DNA to work out the nucleotide sequence of a DNA strand

Question 32

Enzyme assays can be used to do what?

Answer 32

Determine the rate of an enzyme-dependent reaction

Question 33

What does ELISA measure?

Answer 33

The concentration of proteins in a solution