Fragmenting genomic DNA for cloning

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• Fragmenting genomic DNA for cloning
• Random methods are best
• Mechanical shearing sonication, nebulizer
• Nuclease treatment (usually restriction digest)
  4 base cutters, partial digest
• Large fragments better than small, fewer clones
  to get coverage of large genome

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Random fragmentation of genomic
DNA Hydrodynamic shear (physical breakage) --
sonication (vibrating metal probe) --
nebulization (like asthma inhalers) -- passage
through small needle orifice DNA must be
repaired with DNA polymerase after these
treatments Enzymatic breakage -- Restriction
enzyme (4 cutter, partial digest) CviJ (pyGCpy
and puGCpu) -- DNAse I (semi-random cleavage)
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Early library construction
Partial digest Size fractionate Block EcoRI
sites Add linkers Digest with EcoRI Ligate to
lambda Package
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Improved library construction
Partial digest Sau3A (BamHI compatible
ends Phosphatase Ligate to lambda Package
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Improved lambdas for libraries

• More restriction sites
• Sequences for phage RNA polymerase transcription
  (useful for probe synthesis)

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But.

• Cosmids
• BACs
• PACs
• YACs

can be used for cloning larger DNAs using
similar methodsWhy use lambda libraries?
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• Cosmids replicate as high copy number
  plasmids--tend to be unstable, deleting insert
  DNA (to reduce drag on cells)
• BAC and YAC libraries difficult to prepare
• larger-sized DNA more difficult to work with

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Cloning cDNAs

• Prepared by reverse transcription of mRNA
• Eukaryotic mRNAs--lack introns, often show
  variable splicing, cDNAs of these RNAs indicate
  how genes are actually expressed
• Individual mRNA abundance varies widely to
  isolate low abundance mRNAs by cDNA cloning, need
  to make libraries

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• Key points of cDNA cloning
• mRNA source (tissue type) matters a lot
• mRNA must be of high quality (no Rnases.)
• Rare mRNAs can be enriched
• e.g. Subtractive cloning
• hybridize sample cDNA against immobilized
  RNA/cDNA from a driver, clone only those mRNAs
  that are not bound by the driver
• This relies on differential mRNA expression
  between sample and driver mRNA populations

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Gubler/Hoffman method (MC Chapter 11)
1) Synthesize first strand cDNA 2) Second strand
cDNA 3) Methylate cDNA 4) Attach linkers or
adaptors for cloning 5) Fractionate cDNA by size
(select 2-8 kb) 6) Ligate cDNA into
bacteriophage arms
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cDNA libraries
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cDNA synthesis

• Make the first DNA strand from the mRNA template
  using reverse transcriptase
• Remove the RNA
• Make the second DNA strand from the first DNA
  strand

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• Primers for first strand cDNA synthesis
• Oligo dT (binds polyA tails)
• Oligo dT with adaptors (restriction sites)
• Primers linked to a plasmid
• Random primers

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Random priming
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Second strand synthesis early methods
Problem step
Loss of some of the mRNA 5 end
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Second strand synthesis--the Gubler/Hoffman
protocol
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Homopolymer tailing
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But many cDNAs are not full-length--how get only
full-length cDNAs?
Utilize the 5 CAP structure on eukaryotic mRNAs
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cDNA library construction using reverse
transcriptase
cDNA Library Construction Kit (Clontech)
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ESTs Expressed Sequence Tags

• Full length cDNAs hard to get, difficult to scale
  up
• But short cDNA sequences are often useful
• ID and map specific genes
• High throughput allows very fast generation of
  200-300 bp sequences, or ESTs
• Millions of ESTs in database
• Useful in designing microarrays (later)

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cDNA libraries the easy way out
Pre-made cDNA libraries (organisms, tissues,
variable conditions Custom made cDNA libraries
(you supply the mRNA) kits for making your own
cDNA library (See Table 11-6 of Molecular
Cloning for a directory)
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• Library construction
• DNA (entire genome)
• Fragment the DNA
• Clone in lambda phage vector
• mRNA (only the expressed genes)
• First strand cDNA
• Second strand cDNA
• Expressed sequence tags (ESTs)

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Screening libraries for specific genes(finding
the needle in the haystack)

• Isolating individual clones
• Screening by sequence
• Hybridization
• PCR
• Screening by protein structure/biological
  function
• Gene identification--diseases

Course reading 29
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Overview of strategies for cloning genes
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Improved library construction
You want to clone a gene from the human genome
Partial digest Sau3A (BamHI compatible
ends) Phosphatase Ligate to lambda Package
So you follow the protocol for
Orbuy a kit/premade library
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Basic lytic phage life cycle
100s to 1000s of plaques (individual phage
infections)
Lawn of E. coli
Butwhich lambda clone (plaque) has the gene of
interest????
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How many recombinant DNA molecules are required
in a library to get complete coverage of a genome?
p probability of getting a specific piece of DNA
ln(1-p)
N
f fractional size of clone DNA relative to
genome
ln(1-f)
N number of clones needed
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ln(1 - p)
N
ln(1 - f)
p probability of getting a specific piece of
DNA 99
f fractional size of clone DNA relative to
genome 17000 base pairs (lambda capacity) / 3 x
10 9)
N number of clones needed 810,000
ln(1 - 0.99)
810,000
N
ln1 - (1.7 x 104 / 3 x 109)
cDNA cloning this calculation is harder
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Screen by hybridization

• Very fast
• Applicable to a large number of clones
• Can identify clones that are not full length
• But you need to know at least some of the
  sequence of the gene you are after (more on this
  later)

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Design of nucleic acid probes

• Known sequences eg. previously cloned cDNA to
  locate position in genome (identical match exists
  in library--stringent hybridization conditions)
• Probes for non-identical but related sequences
  finding a related gene in another species
  (non-identical match--reduce stringency of
  hybridization)
• Probing for a gene from a sequenced protein eg.
• his-phe-pro-phe-met
• 4) Screen by PCR

make synthetic mixed probe (typically 16-mers)
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guessmers long, degenerate oligo probes

• 40-60 nts, alternative to short, mixed probe
• Codon uncertainty mostly ignored
• Most common codon used
• Increased length improves specificity
• Inosine substitutions at uncertain positions
• Inosine pairs with all 4 bases
• Low stringency hybridizations

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Colony hybridization for ID of clones(like
Southern blotting but without DNA isolation/gel
electrophoresis)
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Plaque-lift hybridization--using a lambda library
Can do this multiple times (replicate experiments)
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Alternative to plating arrayed libraries

• Individual clones of library spotted onto
  membranes in high density arrays (tens of
  thousands of genes)
• Membranes probed as described (a la microarrays)
• Standardizable, centralizable

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Using genomic DNA libraries for mapping
Chromosome walking

• Prior to sequencing
• It is possible to determine the order of clones
  in a contiguous sequence (contig)
• Genes whose general location is known (by
  genetic mapping), but whose function is not
  known, can be found by starting with the genetic
  marker clone and walking away from it

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Chromosome walking how are individual clones in
a genomic library positioned relative to each
other?
The data
The genome assembly
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Chromosome walking

• Probing can be restricted to one direction with
  RNA probes generated from clone ends
• Beware of warping to another chromosome because
  of repetitive sequence probes
• Use YAC and BAC libraries to take larger steps

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Improved lambdas for libraries

• More restriction sites
• Sequences for phage RNA polymerase transcription
  (useful for probe synthesis)

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Expression libraries--alternative to hybridization

• Gene product (protein) is made (by E. coli) and
  detected by variety of methods
• Eukaryotic genes cDNA library is essential (no
  introns, gene size small)
• Screening
• Immunological
• Functional

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Immunological screening
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The plaque lift kind of like a Western blot
Detect antibody with secondary antibody
conjugated to reporter enzyme for visualization
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Functional cloning

• Genetic complementation
• Cloned DNA sequence corrects defect in host
  strain
• Gain of function
• Cloned DNA confers new function to host
• Both of these require cloned DNA to be
  transcribed, translated into functional protein
  in host (eukaryotic protein in E. coli could
  cause problems)
• And you need a good assay for expression!

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Functional complementation shaker gene
Shaker-2 mice have defects in the inner ear, poor
balance, and deafness
The shaker 2 gene encodes myosin XV Mutations in
the human homolog can cause deafness
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• Subtractive cloning
• Remove cDNAs that are common to two sources
• Useful for isolation and detections of
  differentially expressed rare cDNAs
• Example differential expression from
  physiological change
• driver DNA - immobilized
• test cDNA (single stranded) labelled and then
  annealed to driver DNA
• Remaining DNA has no counterpart in the driver
  cells--probe library to locate genes
• Or use the remaining DNA to probe a microarray

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Screening libraries for specific genes(finding
the needle in the haystack)

• Isolating individual clones
• Screening by sequence
• Hybridization
• PCR
• Screening by protein structure/biological
  function
• Gene identification--diseases