week 9_genomic libraries posted
TRANSCRIPT
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BI 312 Week 9 Lecture Overview
DNA libraries
Creation
Uses
Genomic vs. cDNA
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If I want to clone MY FAVOURITE
GENE, how do I get the DNA in the
first place??
PCR
Order it!
Request it from another scientistScreen a genetic library
Some of the following gene library slides were taken from Dr. Mankins lecture found online at http://www.uic.edu/classes/phar/phar331/lecture7/
http://www.uic.edu/classes/phar/phar331/lecture7/http://www.uic.edu/classes/phar/phar331/lecture7/ -
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GENE LIBRARIES
How do you clone one
particular gene from
thousands of genes present
in the genome?
Genome Sizes
E. coli 4.6 million basepairsHousefly 900 million basepairs
Human 3.4 billion basepairs
Pine tree 68 billion basepairs
NOT ASIMPLE
TASK!!!
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The Strategy
Clone everything,then find what you
actually need
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Creating a Genomic Library
1. Isolate chromosomal DNA(Remember you can useANY
tissue source--chromosomal DNAis the same in every cell in an
organism)
2. Digest with a restriction enzyme
3. Digest vector with the samerestriction enzyme
4. Ligate the fragments of
chromosomal DNA with the vector.
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Making a representative library
In order to guarantee that youll be able to find yourgene in the library, you need:
the entire genome to be cloned into the vectors each region of the genome represented at least 2 or 3 times
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Preparing the genomic DNA: partial
digests Time / amount of enzyme in digest is limited so
DNA is NOT cut at every recognition sequence
This will produce longer fragments This will hopefully allow you to clone the full-
length region you desire, and you will requirefewer recombinant clones to cover the library
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Constructing the Library
ligate fragments intovectors
transform ligation
reaction intocompetent cells plate on selective
media only cells that
received arecombinant vectorwill grow; others willbe killed by theselection process
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Constructing the Library
Plate transformed cells on
to agar + antibiotic
Each transformed cell thathas a plasmid will form a
colony; all cells in the
colony have the sameplasmid and cells in
different colonies havedifferent plasmids.
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Choosing your vector the vector you use is determined by the size of the
genome you are working with
prokaryotes have smaller genomes so it is possible tomake a genomic library using a plasmid vector
genes are shorter and inserts can be smaller (5-10 kb) a representative library can be made from a few thousand
recombinants
eukaryotes have larger genomes and require vectors
capable of holding larger inserts genes and inserts are larger, vectors need to hold much larger
fragments of DNA (~20kb)
can use phage, BAC, PAC or YAC
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Making a Phage Library
Fig 7-12 Lodish et al, 4th ed
mix recombinantphages with E. coli
host plate in soft-top agar
isolate phage DNA
from plaques Sometimes 2 different
RE enzymes are used
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Why 2 different RE enzymes ? One of these enzymes must cut the genome frequently (ie a 4-mer; Sau3A) so
that we can obtain a random population of genomic fragments (lots offragments), while the other must cut uniquely in the vector (ie a 6-merenzyme;BamHI) to give specificity(so that we get the gene we are looking for).
SauIIIA and BamHI are the most common enzymes used for this purpose.
BamHI
Cuts vector
SauIIIA
Cuts genomicDNA
GATC
CTAG
GATC
CTAG
G GATCC
CCTAG GG GATCC
CCTAG G
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Cut genomic DNA with SauIIIA
Cut DNA with BamHI
SauIIIA
Cuts genomic
DNA
GATC
CTAG
GATC
CTAG
BamHI
Cuts vector G GATCC
CCTAG G
G GATCC
CCTAG G
Remove replaceable region
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Mix target DNA fragments + arms
Target DNAfragments
CCTAG GGGATC GATCC
CTAG
GATC
CTAG
G
CCTAGGATCC
G
RecombinantDNA
+
arms
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Genomic Libraries: Review
1.
2.
3.4.
5.
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Screening Genomic Libraries:
finding your gene Need to determine which colony is made
up of cells that contain a plasmid with
your gene inserted 4 common methods:
Phenotypic screening
Protein Activity
Immunological screening
DNA hybridization
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Phenotypic Screening
Simplest method
Used when the clonedgene encodes a protein
that changes someproperty of the cells inan obvious and readilyvisible way
For example, the proteinencoded by the clonedgene changes the colourof the transformed cells
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Protein Activity
Used when target gene encodes a proteinthat is an enzyme not native to the host
cells, and that enzyme activity can bereadily detected by eye
Replica-plate on to media that contains
the substrate for the enzyme; onlycolonies containing your gene will act onthe substrate and produce visible products
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Replica-plating
that contains substrate forbiochemical reaction
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Replica-plating
To transfer colonies from one plate toanother maintaining their exact locations,relative to each other and the plate.
Can be accomplished using a sterile fabricpad or nylon filter
Selective/differential media
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Immunological Screening
Using antibodies to screen
the transformed cells Transfer (lift) colonies to a
nylon filter Some of the protein will be
transferred to the filter Hybridize filter with
antibodies that recognizethe protein encoded byyour gene
Positive colonies producecolour
Used when gene of interest is transcribed andtranslated in to protein
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Screening by DNA Hybridization
Requires that the sequence of the target gene isknown
Target DNA (recombinants) is denatured andimmobilized
Design single-stranded probe complementary totarget
Label probe so that only colonies containing thetarget gene light up
Can use radioactivity or colourimetric-basedlabelling strategy
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Screening by DNA Hybridization
Glick & Pasternak, Fig 2.17
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Screening by DNA Hybridization
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Screening by DNA Hybridization
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Screening a Phage Library by DNA
hybridization
Fig. 7-18, Lodish et al. (4th ed.)
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Screening by DNA Hybridization
in general, probes need to be 15-20 nt long in order toguarantee that they are unique (i.e. will bind ONLY to your
gene of interest)
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The Challenge with Eukaryotes Eukaryotes have introns which are removed in the
nucleus after transcription but before translation.
Introns can be longer than exons (as much as 90% of agene!); single gene can be very spread out in thegenome and hard to isolate / analyze
Intron-containing genes cannot be expresed (i.e. madein to proteins) in bacterial cells because prokaryotes
dont have splicing machinery to remove introns
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cDNA
cDNA = complementary DNA Generated from mRNA (so introns have been removed)
using an enzyme called Reverse Transcriptase; makesDNA from an RNA template
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Generating cDNA: first isolate
mRNA Isolate total RNA from eukaryotic
cells
Translated mRNAs contain a
stretch of adenine residues at their3 end (poly A tail)
Use this feature to purify themRNA from the more abundanttRNA and rRNA: bind mRNA tosolid matrix containing shortstretches of thymidine residues(olido dT column)
Lodish et al; Fig 7-14
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Generating a cDNA Library
ds cDNA can thenbe cloned in to avector, andtransformed intoan appropriatehost
cDNA libraries canbe useful for
studying tissue-specific expressionof mRNAs
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Genomic vs. cDNA Libraries
Genomic Source is chromosomal DNA
Include introns
Can be made from ANY tissue source (chromosomalDNA is identical in all cells)
cDNA Source is mRNA
Contains protein-coding sequence (no introns; noregulatory elements associated with genes (e.ipromoters)
Each tissue source gives a different library since
specific mRNAs are expressed in each tissue
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Choosing Your Library
What you want to do with your favourite geneonce you clone it will determine the type oflibrary you should use
Ask the following questions. Do you want tostudy: fragments of the complete genome? DNA sequence?
promoter regions? introns? intron/exon boundaries? protein expression?