MCGB - DNA Hybridisation Flashcards

1
Q

What is meant by the term ssDNA?

A

Single stranded DNA

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2
Q

Outline the stages of Southern blotting.

A
  • digest genomic DNA with restriction enzymes
  • separate DNA fragments by gel electrophoresis
  • transfer DNA fragments to nylon
  • hybridise filter with labelled gene probe
  • detect hybridisation by exposure of filter to x-ray film
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3
Q

What two ways is DNA transferred to the nylon/nitrocellulose filter in a Southern blot?

A

Blotted by capillary action using paper towels, or using electrophoretic transfer (a voltage is applied across the set-up so that negatively charged DNA moves from the gel onto the filter).

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4
Q

Is the DNA on the filter in single or double stranded form?

A

Single-stranded

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5
Q

Give some reasons for using Southern hybridisation.

A
  • to investigate gene structure
  • to investigate gene expansions/triplet repeats
  • to investigate mutations in genetic tests
  • to investigate variation/genetic relationships
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6
Q

Give some limitations of DNA probes in Southern blotting

A
  • probes do not have 100% similarity to target sequence
  • probes do not completely align with target sequence
  • probes do not affect position of target sequence on a gel
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7
Q

What is the Sanger chain termination method?

A

A method of DNA sequencing that uses fluorescent deoxynucleotides.

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