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Hilary Term 1 > The technology underpinning molecular biology: gene analysis > Flashcards

The technology underpinning molecular biology: gene analysis Flashcards

1
Q

How much DNA is there in a human?

A

20^14 copies

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2
Q

How much DNA is there in a bacteria?

A

25^14 per litre

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3
Q

Describe the isolation of cloned DNA

A

alkaline lysis protocol (for plasmid preparation from bacterial cells)

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4
Q

Describe the alkaline lysis protocol

A
  • culture of bacteria containing plasmid vector
  • addition of SDS + NaOH
  • genomic DNA sheared into large, ss linear chunks; plasmids remain supercoiled
  • neutralisation creates tangled mass of linear DNA
  • centrifugation results in pellet of linear genomic DNA + protein and other cell debris
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5
Q

SDS

A
  • sodium dodecyl sulphate
  • ionic detergent
  • pH 12.0-12.5
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6
Q

Describe the Sanger sequencing method

A
  • dNTPs and DNA polymerase added to template to be sequenced
  • in 4 separate tubes, ddA, ddG, ddC and ddT are added
  • OR 1 tube, if fluorescent labelling used
  • products of reaction resolved
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7
Q

Describe ddNTPs

A
  • dideoxynucleotides
  • lack 3’OH
  • block chain extension
  • added at non-saturating concentration
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8
Q

Why are ddNTPs added at a non-saturating concentration

A

chains do not always terminate at the first position

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9
Q

Describe Sanger sequencing analysis under fluorescence

A
  • automated sequencing using fluorescently labelled dNTPs
  • separation of products by size by electrophoresis
  • terminal dideoxynucleotides have different fluors
  • sequence read from primer
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10
Q

Describe Sanger sequencing analysis under radioactive labelling

A

manual sequencing using radiolabelled primer

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11
Q

Describe Sanger sequencing analysis using lasers

A

automated sequencers use capillary electrophoresis and laser detection of products
- laster projector shot t +ve end, where the detector is
- gives a chromatogram and sequence readout

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12
Q

How is sequencing used to determine gene structure?

A
  • comparison between cDNA and genomic clone sequences
  • promoters, introns and exons can be determined
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13
Q

How is gene copy number estimated?

A
  • agarose gel electrophoresis
  • restriction enzyme digests genomic DNA samples
  • produces smears of 1000s of different sized fragments
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14
Q

Describe Southern blot analysis

A
  • unlabelled genomic DNA cut with restriction enzyme
  • DNA fragments separated by agarose gel electrophoresis
  • denaturation with NaOH
  • agarose gel placed atop a sponge in alkali solution, with nitrocellulose paper and a stack of paper towels on top
  • capillary blotting
  • remove nitrocellulose paper with tightly bound DNA, and fix to paper by baking/UV treatment
  • labelled DNA probe hybridised to separated DNA on nitrocellulose
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15
Q

capillary blotting

A

blotting onto nitrocellulose paper

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16
Q

Describe the isolation of cloned DNA from a vector

A
  • plasmid DNA prepared by alkaline lysis method
  • cut with EcoRI (same restriction enzyme used in cloning)
  • fragments separated by size using agarose gel electrophoresis
  • relevant bands cut out
  • DNA extracted
  • cloned fragment can be purified to make a probe template for labelling reaction
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17
Q

Describe estimation of gene copy number

A
  • nitrocellulose paper from Southern blotting experiment hybridised with a radiolabelled probe
  • autoradiography
  • count band no in each sample
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18
Q

autoradiography

A

exposure to X-ray film

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19
Q

What are weaker bands indicative of?

A

divergent copies of a gene

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20
Q

Describe determination of chromosomal localisation

A

FISH

21
Q

FISH - the basics

A
  • fluorescent in situ hybridisation
  • determines chromosomal localisation
22
Q

FISH - the specifics

A
  • drop metaphase cells onto glass slide
  • sample fixed and permeabilised
  • DNA denatured
  • hybridisation probes labelled with fluorescent dye added
  • visualise by fluorescence microscopy
23
Q

Why aren’t radioactive probes used in FISH

A
  • insufficient resolution
  • may damage chromosomes
24
Q

How to find out when and where a gene is expressed?

A
  • blot-based
  • in situ
25
Q

Describe Northern blot analysis

A
  • RNA of various tissues purified
  • RNA samples loaded into agarose gel wells
  • samples separated by gel electrophoresis
  • blotted onto nitrocellulose filter
  • filter exposed to labelled hybridisation probe
26
Q

Compare Southern and Northern blot analysis

A
  • same principles, but Southern blotting does DNA, where Northern blotting does RNA
  • in Northern blotting, fragmentation step is not required
  • unhybridised probe washed away
  • autoradiography reads tissue-specific transcripts
27
Q

Describe the probe used in Northern blotting

A
  • can be DNA as in Southern blotting
  • DNA:RNA hybrids
28
Q

What does Northern blotting provide>

A

information on mRNA levels in different tissue samples, and mRNA size

29
Q

Describe in situ detection of gene expression

A
  • in situ hybridisation
  • determines exactly which cells accumulate mRNA of interest
30
Q

Describe the preparation of tissue for in situ hybridisation

A
  • gradient set up of water -> 70% alcohol -> 95% alcohol -> absolute alcohol -> histoclear
  • tissue fixed, embedded, sectioned, stained and mounted
  • tissue ready for analysis
31
Q

Describe the probe of in situ hybridisation

A
  • RNA probe labelled digoxigenin
  • antisense so it will hybridise to mRNA
  • ss for sensitivity
  • T7 polymerase (from bacteriophage T7)
32
Q

How a ss probe made?

A
33
Q

Describe the cDNA of interest in in situ hybridisation

A
  • inverted orientation
  • behind T7 promotor
34
Q

digoxigenin

A
  • DIG-modified nucleotide
  • rare plant steroid molecule
  • highly antigenic
  • immune tag
  • UTP-DIG
35
Q

Describe the alternative probe for in situ gene expression analysis

A
  • fluorescence
  • requires RNA FISH
36
Q

Describe detection of a DIG labelled probe

A
  • uses alkaline phosphatase, conjugated to anti-DIG antibody
  • forms insoluble purple precipitate at sites of alkaline phosphatase activity
  • visualised by microscopy
  • each cell with purple stain contains mRNA of interest
37
Q

Describe analysis of protein accumulation profiles

A
  • blot-based
  • in situ
  • requires an antibody
38
Q

Describe antibodies for protein detection

A
  • dual-antibody detection systems allows for signal amplification
  • single detection reagent (secondary antibody conjugate) for multiple protein targets
39
Q

Why is signal amplification good?

A

improves sensitivity

40
Q

How is the primary antibody synthesised?

A
  • using cloned gene to synthesis protein
  • tag with hexahistidine
41
Q

Describe the purification of His tagged proteins

A
  • hexahistidine tag on protein of interest will bind to nickel column
  • cells with expressed protein lysed
  • cell extract loaded into column
  • column washed, releasing unbound proteins
  • nickel bound bead attaches 6-His and expressed protein
  • elute tagged protein
  • protein is purified
  • immunise animal to make antibody
42
Q

Describe Western blot analysis

A
  • uses polyacrylamide gel
  • proteins blotted to membrane using electroblotting
  • probe blot with primary, then secondary antibodies
  • detect alkaline phosphatase using chromogenic substrate
  • assess presence and relative abundance
43
Q

Why is polyacrylamide gel used in Western blot analysis?

A

better resolution than agarose gels

44
Q

Why is electroblotting used for Western blotting, rather than capillary blotting?

A

polyacrylamide matrix is much tighter than agarose

45
Q

Describe electro blotting

A
  • nitrocellulose membrane placed under gel
  • filter paper either side
  • cathode one side and anode the other
46
Q

Describe the analysis of relative abundance in Western blotting

A

proportional to band intensity and protein size

47
Q

Describe protein and RNA localisation patterns in situ

A
  • immunohistochemistry
48
Q

Compare western blotting and immunohistochemistry

A
  • very similar
  • immunohistochemistry uses tissue section instead of gel blot

Hilary Term 1 (61 decks)

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