The technology underpinning molecular biology: gene analysis Flashcards
How much DNA is there in a human?
20^14 copies
How much DNA is there in a bacteria?
25^14 per litre
Describe the isolation of cloned DNA
alkaline lysis protocol (for plasmid preparation from bacterial cells)
Describe the alkaline lysis protocol
- 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
SDS
- sodium dodecyl sulphate
- ionic detergent
- pH 12.0-12.5
Describe the Sanger sequencing method
- 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
Describe ddNTPs
- dideoxynucleotides
- lack 3’OH
- block chain extension
- added at non-saturating concentration
Why are ddNTPs added at a non-saturating concentration
chains do not always terminate at the first position
Describe Sanger sequencing analysis under fluorescence
- automated sequencing using fluorescently labelled dNTPs
- separation of products by size by electrophoresis
- terminal dideoxynucleotides have different fluors
- sequence read from primer
Describe Sanger sequencing analysis under radioactive labelling
manual sequencing using radiolabelled primer
Describe Sanger sequencing analysis using lasers
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
How is sequencing used to determine gene structure?
- comparison between cDNA and genomic clone sequences
- promoters, introns and exons can be determined
How is gene copy number estimated?
- agarose gel electrophoresis
- restriction enzyme digests genomic DNA samples
- produces smears of 1000s of different sized fragments
Describe Southern blot analysis
- 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
capillary blotting
blotting onto nitrocellulose paper
Describe the isolation of cloned DNA from a vector
- 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
Describe estimation of gene copy number
- nitrocellulose paper from Southern blotting experiment hybridised with a radiolabelled probe
- autoradiography
- count band no in each sample
autoradiography
exposure to X-ray film
What are weaker bands indicative of?
divergent copies of a gene
Describe determination of chromosomal localisation
FISH
FISH - the basics
- fluorescent in situ hybridisation
- determines chromosomal localisation
FISH - the specifics
- drop metaphase cells onto glass slide
- sample fixed and permeabilised
- DNA denatured
- hybridisation probes labelled with fluorescent dye added
- visualise by fluorescence microscopy
Why aren’t radioactive probes used in FISH
- insufficient resolution
- may damage chromosomes
How to find out when and where a gene is expressed?
- blot-based
- in situ
Describe Northern blot analysis
- 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
Compare Southern and Northern blot analysis
- 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
Describe the probe used in Northern blotting
- can be DNA as in Southern blotting
- DNA:RNA hybrids
What does Northern blotting provide>
information on mRNA levels in different tissue samples, and mRNA size
Describe in situ detection of gene expression
- in situ hybridisation
- determines exactly which cells accumulate mRNA of interest
Describe the preparation of tissue for in situ hybridisation
- gradient set up of water -> 70% alcohol -> 95% alcohol -> absolute alcohol -> histoclear
- tissue fixed, embedded, sectioned, stained and mounted
- tissue ready for analysis
Describe the probe of in situ hybridisation
- RNA probe labelled digoxigenin
- antisense so it will hybridise to mRNA
- ss for sensitivity
- T7 polymerase (from bacteriophage T7)
How a ss probe made?
Describe the cDNA of interest in in situ hybridisation
- inverted orientation
- behind T7 promotor
digoxigenin
- DIG-modified nucleotide
- rare plant steroid molecule
- highly antigenic
- immune tag
- UTP-DIG
Describe the alternative probe for in situ gene expression analysis
- fluorescence
- requires RNA FISH
Describe detection of a DIG labelled probe
- 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
Describe analysis of protein accumulation profiles
- blot-based
- in situ
- requires an antibody
Describe antibodies for protein detection
- dual-antibody detection systems allows for signal amplification
- single detection reagent (secondary antibody conjugate) for multiple protein targets
Why is signal amplification good?
improves sensitivity
How is the primary antibody synthesised?
- using cloned gene to synthesis protein
- tag with hexahistidine
Describe the purification of His tagged proteins
- 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
Describe Western blot analysis
- 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
Why is polyacrylamide gel used in Western blot analysis?
better resolution than agarose gels
Why is electroblotting used for Western blotting, rather than capillary blotting?
polyacrylamide matrix is much tighter than agarose
Describe electro blotting
- nitrocellulose membrane placed under gel
- filter paper either side
- cathode one side and anode the other
Describe the analysis of relative abundance in Western blotting
proportional to band intensity and protein size
Describe protein and RNA localisation patterns in situ
- immunohistochemistry
Compare western blotting and immunohistochemistry
- very similar
- immunohistochemistry uses tissue section instead of gel blot
