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Molecular Biology and Medicine > DNA analysis and nucleic acid probes > Flashcards

DNA analysis and nucleic acid probes Flashcards

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1
Q

What is a shotgun approach?

A

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

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2
Q

What is a genomic library?

A

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

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3
Q

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

A

A screening procedure

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4
Q

How else can DNA be cloned?

A

Phages

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5
Q

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

A
  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
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6
Q

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

A

Hybridisation

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7
Q

What is important to remember when comes to restriction enzymes?

A

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

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8
Q

Why is a cDNA library important?

A

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

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9
Q

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

A

Brain and liver

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10
Q

What does BAC stand for?

A

Bacterial artificial chromosome

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11
Q

Can a BAC be used as a vector?

A

Yes

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12
Q

What is a BAC?

A

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

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13
Q

A large insert size minimises what?

A

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

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14
Q

BAC are usually stored in what?

A

Usually stored in multiwell plates, one clone per well

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15
Q

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

A

mRNA extracted from cells

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16
Q

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

A

Primer

Reverse transcriptase derived from the retrovirus

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17
Q

dsDNA is called what?

A

complementary DNA or cDNA

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18
Q

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

A

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

19
Q

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

A

cDNA there are no introns

20
Q

cDNA is good for studying what?

A

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

21
Q

What is nucleic acid hybridisation?

A

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

22
Q

Denaturation is important to do what?

A

Separate the DNA strands to ssDNA

23
Q

How is denaturation done?

A

Chemicals or heat

24
Q

Where does hybridisation take place?

A

Special filter - probe molecules are incubated with the filter

25
Q

The probe DNA hybridises what?

A

base pairs with complimentary DNA on the filter

26
Q

What is done with the excess DNA in hybridisation?

A

Rinsed off

27
Q

What is done with the filter after hybridisation is completed?

A

Filter laid on film to detect radioactivity (autoradiography)

28
Q

What is the autoradiography compared to?

A

Master plate to find colonies carrying the desired gene

29
Q

Name several techniques to analyse DNA

A
  1. Gel electrophoresis
  2. Restriction analysis
  3. Southern Blotting
  4. Exploiting differences in gene sequences that affect restriction sites (RFLPs)
30
Q

What is Gel electrophoresis used for?

A

Separating various sizes of macromolecules

31
Q

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

A

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

32
Q

What is an example of Genomics?

A

Human Genome Project

33
Q

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

A

Combine the gel electrophoresis result with nucleic acid hybridisation

34
Q

When was Southern Blotting developed?

A

1975

35
Q

Who developed southern blotting?

A

E.M Southern

36
Q

Southern Blotting is used for what?

A

Detection and analysis of particular sequences using nucleic acid hybridisation

37
Q

What is Southern Blotting useful for?

A

Comparing DNA of different individuals or even species

38
Q

Describe the process of Southern Blotting

A

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

39
Q

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

A

non-coding DNA

40
Q

Southern Blotting found that non-coding DNA showed what?

A

Showed differences in restriction patterns just like alleles

41
Q

What does RFLPs stand for?

A

Restriction Fragment Length Polymorphisms

42
Q

RFLPs are good for what?

A

Linkage maps

43
Q

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

A

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

Molecular Biology and Medicine (9 decks)

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