BIO2015: Lecture 3

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  • Created by: LMoney
  • Created on: 12-05-14 09:50
why is cloning all of genomic DNA of higher organisms into plasmid vectors not practical?
relatively low transformation efficiency of E.coli and small number of transformed that can be grown on typical culture plate
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what are bacteriophage?
viruses that infects and replicates within bacteria- have no such limitations as plasmids
2 of 50
what is a genomic library?
Collection of clones that includes all DNA sequences of given species
3 of 50
what do bacteriophage λ undergo following infection of E. coli?
either lytic replication or lysogeny
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what does lytic replication of bacteriophage λ result in?
cell death of E.coli and formation of clear plaques on cell lawn
5 of 50
what are the heads and tails of bacteriophage λ?
systems of self assembling proteins
6 of 50
addition of DNA containing COS sites at correct spacing (43-53kb) allows what?
spontaneous formation of complete, functional λ phage- basis of in vitro packaging system
7 of 50
preassembled λ head and (blank) of λ DNA combined?
concatomer
8 of 50
which proteins promote filling of λ head with DNA between COS sites?
Nu1 and A proteins
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what is contained in preassembled λ head?
a-genome (1 copy)- preassembled λ tail is then attached because λ attaches only to filled head
10 of 50
what mode of replication is used in bacteriophage lambda DNA?
rolling circle mode at the COS site
11 of 50
what cleaves at COS sites?
endonuclease A- Lambda proteins assembled DNA packaged in phage head (upper limit 53 kb)- combined give infective particles
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what is the central stuffer fragment in y-phage vectors?
central stuffer fragment contains genes used in the lysogenic phase of the life cycle - i.e. to cause the phage to integrate into the host genome, and to maintain the integrated state- it is replaced by the DNA to be cloned
13 of 50
which part of the life cycle do y-phage cloning vectors undergo?
the lytic part of the normal phage life cycle
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why is there a restricted size range of fragments that can be accepted?
When replacing the stuffer fragment, the DNA fragments to be cloned must allow functional phage to be produced
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Vectors designed for cloning genomic DNA generally accept fragments in what range?
12-20 kbp- vectors designed for cloning cDNA accept fragments in the range 0-10kbp
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what is the efficiency of host infection (transfection) not dependent on?
insert size, provided that phage in correct size range results
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which have linear DNA, plasmid or phage vectors?
phage vectors, plasmid are circular
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In a phage vector, is the DNA to be cloned inserted or does it replace host DNA?
replaces it
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which retains transfection efficiency over size ranges- plasmid or phage vectors?
phage- transformation falls off rapidly with insert size in plamids
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which perform better for larger inserts phage or plasmid vectors?
phage (>5kbp), plasmid (
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which are easier to handle?
bacterial clones containing plasmids- Phage clones more difficult to handle- must be plated onto bacterial lawns
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for which type of vector is the density of clones limited?
plasmid vectors- 1000 per plate- phage vectors can have >10,000 per plate
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which is easier to prepare- plasmid or phage DNA?
Plamid
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which are better for large libraries phage vectors or plasmid vectors?
phage
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what are cDNA libraries prepared from?
isolated mRNAs
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how is cDNA produced?
Reverse transcriptase makes DNA copy from RNA template- requires primer- linkers may be added to ends of DNA to help join to vector
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genomic libraries are all that is necessary for what type of organism?
prokaryotes- expressed sequences and encoded proteins can be easily deduced
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what do cloned fragments include in genomic libraries?
both transcribed and non-transcribed regions (i.e. include promoters)- include introns and axons
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what do cloned fragments not include in genomic libraries?
poly(A) ‘tails’
30 of 50
does the genomic library differ if different tissues or samples from different developmental stages are used?
no, remains the same
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in genomic libraries where is the cloned DNA from?
directly from genome
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in cDNA libraries where are the cloned DNA fragments obtained from?
copies of mRNA sequences
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both types of library (genomic and cDNA) are needed for what type of organism?
eukaryote- presence of large amounts of intergenic DNA and complex structure of genes hinders gene characterization
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in cDNA libraries cloned fragments contain what?
only transcribed regions, exons only
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in cDNA libraries, do the cloned fragments have poly(A) tails?
yes, and other mRNA modifications
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are samples from different developmental stages used in cDNA libraries?
yes, libraries made from different tissues and/or developmental stages of organism- differ because different genes transcribed into mRNA
37 of 50
in genomic and cDNA libraries which need more and which need less clones?
genomic need more (>10^6), cDNA need fewer (
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in screening a library, how are E.Coli colonies or plaques that contain DNA of interest detected?
Based on: Enzyme activity or protein or DNA sequence
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why is detection of gene product often impractical?
foreign gene may not be expressed or product may be inactive
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how else can screening be carried out?
Antibodies are used to detect a specific protein in complex mix- can also be used to measure amount of a protein
41 of 50
what are antibodies produced by?
B cells
42 of 50
what happens when you inject immunogen into animal?
animal produces antibodies that bind specifically to immunogen
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what is the procedure for screening with antibodies?
1)Plate out library on agar 2)Transfer to filter 3)Lyse cell to release protein • Add inert protein (e.g. bovine serum albumin to block non-specific binding) • Incubate with radioactive antibody • Expose to X-ray film • Radioactive antibody blackens
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what is nucleic acid hybridisation used for?
used to detect specific DNA or RNA sequence in complex mix- can also be used to measure amount of sequence
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what is the mechanism for nucleic acid hybridisation?
complementary sequences bind to one another by base pairing
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where can nucleic acid hybridisation take place?
1) In solution 2) With target DNA attached to filter (nylon or nitrocellulose)
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in library screening using a DNA probe, what can you use as a probe?
1) Most commonly, a sequence that is related to the required gene 2) a similar sequence from the same or another organism 3) cDNA to identify genomic DNA clone
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what is the process of screening with a DNA probe?
1) plate out library on agar 2) transfer to filter 3) lyse cells and denature DNA with alkali 4) add salmon sperm DNA to block non-specific binding 5) incubate with radioactive probe 6) expose to x-ray film 7) radioactive probe blackens film
49 of 50
describe differential screening
1) make cDNA library from liver 2) hybridise with labeled total cDNA from liver and muscle- clones hybridizing only to liver cDNA are differentially expressed
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Card 2

Front

what are bacteriophage?

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viruses that infects and replicates within bacteria- have no such limitations as plasmids

Card 3

Front

what is a genomic library?

Back

Preview of the front of card 3

Card 4

Front

what do bacteriophage λ undergo following infection of E. coli?

Back

Preview of the front of card 4

Card 5

Front

what does lytic replication of bacteriophage λ result in?

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