DNA analysis and nucleic acid probes

Question 1

What is a shotgun approach?

Answer 1

A mixture of fragments from the whole genome, it’s called a shotgun approach

Question 2

What is a genomic library?

Answer 2

The set of 1000’s of recombinant clones, each carrying copies of a particular fragment from the initial genome is called a genomic library

Question 3

What must be done to the genomic library to find the colony with the gene of interest?

Answer 3

A screening procedure

Question 4

How else can DNA be cloned?

Answer 4

Phages

Question 5

Baceteriophage lambda is one of the most widely used for several reasons. What are they?

Answer 5

1. Molecular genetics well known
2. Can hold larger amounts of DNA than plasmids
3. More efficient than transformation - transfer by transduction
4. Recombinant DNA is packaged in phage heads in vitro
5. Phage infects bacteria
6. Plaques form where bacteria have lysed

Question 6

The plaques formed from bacteriophages can be screened for the gene of interest using what process?

Answer 6

Hybridisation

Question 7

What is important to remember when comes to restriction enzymes?

Answer 7

They don’t respect gene boundaries - so some genes in the library may be spread over two or more clones

Question 8

Why is a cDNA library important?

Answer 8

Because it will be limited to the genes that are being transcribed in the starting cells

Question 9

cDNA libraries are important for studying gene expression in particular types of cells e.g?

Answer 9

Brain and liver

Question 10

What does BAC stand for?

Answer 10

Bacterial artificial chromosome

Question 11

Can a BAC be used as a vector?

Answer 11

Yes

Question 12

What is a BAC?

Answer 12

A BAC is a large plasmid that has been trimmed down and can carry a large DNA insert (100-300 kb)

Question 13

A large insert size minimises what?

Answer 13

Large insert size minimises number of clones needed to make up genomic library

Question 14

BAC are usually stored in what?

Answer 14

Usually stored in multiwell plates, one clone per well

Question 15

A more limited kind of gene library can be developed by starting with what?

Answer 15

mRNA extracted from cells

Question 16

The process of a retrovirus requires what to make a gene library?

Answer 16

Primer

Reverse transcriptase derived from the retrovirus

Question 17

dsDNA is called what?

Answer 17

complementary DNA or cDNA

Question 18

What do you need to do to the cDNA to make create a library?

Answer 18

cDNA is modified by adding restriction sites to each end and then inserting into vector DNA

Question 19

If you are only interested in the coding sequence of a gene do you use cDNA or mRNA and why?

Answer 19

cDNA there are no introns

Question 20

cDNA is good for studying what?

Answer 20

Studying specialised functions of cells and studying gene expression at different stages of development of an organism

Question 21

What is nucleic acid hybridisation?

Answer 21

Nucleic acid hybridisation consists of base pairing between the gene and a complementary sequence on another nucleic acid molecule – nucleic acid probe
This short ss nucleic acid can either be RNA or DNA
If we know at least part of the sequence of our gene of interest can then synthesise a probe complementary to it
Probe can be traced by radioactive labelling or fluorescent marker
The probe will bond specifically to complementary ssDNA of the gene of interest

Question 22

Denaturation is important to do what?

Answer 22

Separate the DNA strands to ssDNA

Question 23

How is denaturation done?

Answer 23

Chemicals or heat

Question 24

Where does hybridisation take place?

Answer 24

Special filter - probe molecules are incubated with the filter

Question 25

The probe DNA hybridises what?

Answer 25

base pairs with complimentary DNA on the filter

Question 26

What is done with the excess DNA in hybridisation?

Answer 26

Rinsed off

Question 27

What is done with the filter after hybridisation is completed?

Answer 27

Filter laid on film to detect radioactivity (autoradiography)

Question 28

What is the autoradiography compared to?

Answer 28

Master plate to find colonies carrying the desired gene

Question 29

Name several techniques to analyse DNA

Answer 29

1. Gel electrophoresis 2. Restriction analysis 3. Southern Blotting 4. Exploiting differences in gene sequences that affect restriction sites (RFLPs)

Question 30

What is Gel electrophoresis used for?

Answer 30

Separating various sizes of macromolecules

Question 31

Why do some molecules move through a gel further than others?

Answer 31

The smaller the DNA fragments the easier it is for them to get through the agarose pores and therefore travel furthest on the gel.

Question 32

What is an example of Genomics?

Answer 32

Human Genome Project

Question 33

What do you do when your gel has just a smear because the bands are all too close together?

Answer 33

Combine the gel electrophoresis result with nucleic acid hybridisation

Question 34

When was Southern Blotting developed?

Answer 34

1975

Question 35

Who developed southern blotting?

Answer 35

E.M Southern

Question 36

Southern Blotting is used for what?

Answer 36

Detection and analysis of particular sequences using nucleic acid hybridisation

Question 37

What is Southern Blotting useful for?

Answer 37

Comparing DNA of different individuals or even species

Question 38

Describe the process of Southern Blotting

Answer 38

First, samples are digested with restriction enzymes Restriction fragments are separated by electrophoresis Blotting – an alkaline solution is pulled up through the gel and through a sheet of nitrocellulose paper on top of gel DNA is transferred to the paper, denaturing to ssDNA which sticks to paper Bands on paper exactly same as on the gel Paper blot treated with radioactive probe complementary to the sequence of interest Probe attaches by base-pairing to fragments of complementary sequence Perform autoradiograpy to reveal bands containing DNA that bound to probe

Question 39

Southern Blotting of restriction fragments proved useful in analysis of what?

Answer 39

non-coding DNA

Question 40

Southern Blotting found that non-coding DNA showed what?

Answer 40

Showed differences in restriction patterns just like alleles

Question 41

What does RFLPs stand for?

Answer 41

Restriction Fragment Length Polymorphisms

Question 42

RFLPs are good for what?

Answer 42

Linkage maps

Question 43

What part did RFLPs have to play in the human genome project?

Answer 43

Greatly increased the number of markers that could used in mapping the human genome