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2014 Genetics Topic 3: RG > L3 > Flashcards

L3 Flashcards

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Biology 101 flashcards
1
Q

true or false, libraries can be constructed containing all the genes in an organism or all the genes expressed in a particular tissue

A

true

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2
Q

what kind of libraries do BAC vectors help construct

A

genomic libraries

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3
Q

what is a genomic library

A

a set of clones containing all of the genomic/ chromosomal DNA of an organism

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4
Q

what are the requirements for contructing a genomic library with a BAC vector

A
  • DNA can be from any cell

- even representation of genome is crucial

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5
Q

describe the preparation of genomic library

A
  • cut the DNA with blunt cutting RE, phosphotase (stops circularisation)
  • prepare high MW DNA
  • random shear, blunt ends
  • gel purify large fragments
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6
Q

what is a cDNA library

A

set of clones containing DNA copies of all mRNAs expressed in a certain cell type of an organism

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7
Q

how are cDNA libraries constructed

A
  1. ) isolate population of mRNA from cells (oligo-dT column)
  2. ) anneal oligo-dT primer, copy to DNA with reverse transcriptase
  3. ) treat with RNAseH (attacks RNA moiety of RNA-DNA hybrid)
  4. ) treat with DNA pol 1 to make dsDNA
  5. ) clone into plasmids or lambda vectors
    - can clone blunt, or ligate linkers
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8
Q

true or false, early cDNA clones often incomplete at 5’ ends, premature termination of RT, loss of bases at 5’ end during polishing, new method replace cap with a known oligo

A

true

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9
Q

Describe BAC genomic library

A
  • source is chromosomal DNA
  • all genes in an organism
  • has introns, promoters, repeat sequences, TE (transposable elements)
  • higher sequence complexity
  • cloned as large fragments (in BACs, 100-150 kb)
  • can be multiple (or partial) genes per clone
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10
Q

describe plasmid cDNA library

A
  • source is mRNA
  • genes expressed in source tissue
  • has coding regions and adjacent transcribed sequences only
  • lower complexity (<1.5% in human)
  • small inserts (1-2 kb on average) in plasmid or lambda vectors
  • always one gene per clone
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11
Q

true or false, to a large extent PCR amplification from either genomic DNA or cDNA has replaced construction and screening

A

true

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12
Q

what is the “catch 22”

A
  • means that with PCR it requires that you know enough about your target gene so that you can design primers, however even today if you are looking for a totally new gene function a library screening approach is often still taken
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13
Q

true or false, making libraries is fairly straight forward, the real problem is finding the gene

A

true

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14
Q

what are the 3 common ways of identifying genes in a library

A
  • probes
  • antibodies
  • complementation
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15
Q

how big is the mammalian genome

A

3 Million kb

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16
Q

what is the average BAC insert in a mammalian genome

A

100 kb

17
Q

how large is a single coverage library for a mammalian genome

A

30 000 clones

18
Q

how many clones does a representative library of a mammalian genome have

A

150 000 clones

19
Q

what is the expected frequency for a single copy gene of in a mammal

A

1/30 000 BAC clones

20
Q

true or false, although once you have identified the right BAC, the actual gene still needs to be subcloned

A

true

21
Q

how large is the average human gene

A

14 kb

22
Q

in a mammalian cell how much mRNAs

A

2 times 10^5 mRNAs

23
Q

representative library of cDNA of mammal

A

one gene per clone, 1 million clones

24
Q

true or false, in mammals the abundance of mRNA varies from 1 copy per 10 cells, up to 100 000 per cell

A

true

25
Q

what is the expected frequency of cDNA library of mammalian cell

A

50% to 10-6

26
Q

true or false, in mammalian cDNA library, the abundance can be zero if gene not expressed in the starting tissue; gene youre looking for must be expressed in the starting tissue

A

true

27
Q

when do you use a cDNA library

A
  • when looking for a coding region, source is mRNA, gene must be expressed in the source tissue
28
Q

true or false, use of cDNA library allows expression of the genes in E.coli because there are no introns

A

true

29
Q

true or false, the cDNA library needs to be constructed from a tissue where gene is highly expressed, if you don’t know this information, you need to use the genomic library

A

true

30
Q

true or false, need to use a genomic library when the promoter, polyadenelation signal or introns are sought, rather than just the coding region

A

true

31
Q

how to make a probe; method 1: colony or plaque colonisation using a labelled probe

A

the clone carrying the gene of interest is identified by probing a genomic library (looking for promoter). In this case made by cloning genes in a fosmid vector, with DNA or RNA known to be related to the desired gene.
A radioactive probe hybridises with any recombinant DNA incorporating a matching DNA sequence, and the position of the clone having the DNA is revealed by autoradiography

32
Q

true or false, DNA can anneal if there is > 70% DNA sequence of homology over 100 bases

A

true

33
Q

true or false, can adjust the stringency of hybridisation to get the kind of matches you need by changing the temperature ( or salt ).

A

true

34
Q

true or false, annealing at 85 degrees in standard salt conditions will mean only perfect matches will stick

A

true

35
Q

true or false, annealing at 65 degrees in standard salt means up to 20% mismatch will still anneal

A

true

36
Q

true or false, we can manipulate stringency by manipulating salt concentration and temperature

A

true

37
Q

how to design oligonucleotide probes from protein sequence

A

A short sequence of a protein is used to design a set of redundant oligonucleotides for use as a probe to recover the gene that encoded the protein, one of the set of probes will be a perfect match for the gene

  • inserting the least degenerate 20 base region of codon combination, this must be present in the hybrid probe to ensure that it will stick to the target mRNA
38
Q

Describe method 2: Antibody detection of an expressed protein

A
  • purify protein
  • inject purified protein into rabbit/mouse/chicken and purify the antibodies
  • extract their blood
  • construct an expression library (cDNA clones behind promoter, often as fusion protein)
  • in lambdagt11- lambda insertion vector with lacZ gene- 1/6 of inserts express a fusion protein
  • specificity resides in specificity of antibody binding
  • use of secondary antibodies amplifies signal
39
Q

describe method 3: Complementation of E.coli or yeast mutants

A
  • has been widely used to clone conserved “core functions” such as genes involved in amino acid biosynthesis, transcription, translation, transport, cell division
  • often needs mutation in either E.coli or yeast (and thus a selection for a functional gene)
  • more difficult library construction- need to express complete functional copies of active proteins in E.coli or yeast, ie whole cDNA insert cloned into expression vector

2014 Genetics Topic 3: RG (10 decks)

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