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a' Library screening: plaque hybridization

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The gene from a genomic library using the cDNA as a probe ... This is a clear area caused by the lysis. of bacteria on the lawn of E. coli. ... – PowerPoint PPT presentation

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Title: a' Library screening: plaque hybridization


1
10. Isolating Genes
a). Library screening plaque hybridization b).
Probes for library screening i). Starting with a
protein synthetic oligonucleotide antibody ii
). Starting with mRNA differential cDNA library
screening expression screening
2
  • Plaque hybridization
  • This is a general technique required for a
    number of
  • specific approaches for isolating cDNA
    or genomic clones
  • Generally, one starts by
  • 1). Isolating a cDNA sequence from a cDNA
    library, then
  • 2). The gene from a genomic library using the
    cDNA as a probe
  • Information gained from cDNA and genomic clones
  • 1). cDNA clones provide the amino acid sequence
    of the
  • full-length protein, unencumbered by intron
    sequences
  • 2). Genomic clones provide the control regions
    and are
  • required for searching for mutations
  • Library screening four experimental approaches
  • Starting with a protein
  • 1). Synthetic oligonucleotide - plaque
    hybridization
  • 2). Antibody - variation of plaque
    hybridization
  • Starting with mRNA

3
  • Library screening
  • plaque hybridization
  • plate phage library on lawn of E. coli (bacteria
    gtgtgt phage)
  • plaques form as a consequence of a spreading
    lytic infection
  • starting with a single phage-infected bacterial
    cell
  • each phage plaque is a clone of identical
    recombinant phage
  • prepare replica of phage plaques and hybridize
    DNA with probe

E. coli lawn is grown on agar plate and
then overlayered with the recombinant phage
library.
Wherever a single bacteriophage particle infects
a bacteria cell, a plaque will form. This is a
clear area caused by the lysis of bacteria on the
lawn of E. coli.
A replica of the agar plate is made on
a nitrocellulose sheet - the DNA is denatured and
adheres to the nitrocellulose.
The nitrocellulose is hybridized with a
labeled DNA probe (such as an oligonucleotide)
and the nitrocellulose is exposed to X-ray film.
X-ray film
spot on film indicates a plaque containing DNA of
interest
4
(No Transcript)
5
  • How does one isolate a gene for an inherited
    disorder?
  • Start with a candidate protein
  • DNA protein
  • If a protein candidate has been identified for a
    genetic
  • disease it can be used to make a probe to screen
    for the gene
  • 1. oligonucleotide probe
  • purify the protein of interest
  • partially sequence the protein
  • find a region having amino acids with the fewest
    possible codons
  • predict a DNA sequence that could represent a
    gene region
  • encoding a portion of the protein
  • synthesize a set of degenerate oligonucleotides
    for that region
  • hybridize the labeled oligonucleotide to the
    phage library

MET.GLU.PHE.TYR.ILE.CYS.GLN.LYS amino
acids AUG.GAA.UUU.UAU.AUU.UGU.CAA.AAA G C
C C C G G all possible
A oligonucleotides 1 X 2 X 2 X
2 X 3 X 2 X 2 X 2 192-fold degenerate

6
  • 2. antibody probe
  • purify the protein of interest
  • make an antibody to that protein
  • construct cDNA library to express recombinant
    proteins in E. coli
  • use the antibody to detect the protein being
    made from the cloned
  • cDNA encoded by the recombinant phage in E.
    coli using plaque
  • hybridization method modified for antibody
    probes

bacterial promoter and Shine-Dalgarno sequence
left arm
right arm
human cDNA insert
7
  • How does one isolate a gene for an inherited
    disorder?
  • Start with a candidate mRNA
  • DNA mRNA
  • mRNA candidates can be identified by comparing
    mRNA
  • populations between normal and abnormal tissues,
    or by
  • looking for a specific function encoded by the
    mRNA
  • 1. differential cDNA library screening
  • prepare duplicate plaque replica plates
  • hybridize one with a labeled cDNA probe made to
    all the mRNAs
  • in the normal cell and hybridize the other
    (duplicate) with the
  • corresponding probe to the abnormal cell
  • differences in cell function should be reflected
    by differences
  • in the mRNA populations
  • any plaques showing differential hybridization
    are candidates

no hybrid- ization to this plaque
differentially expressed clone
hybridization with cDNA probe from
normal cells
hybridization with cDNA probe from
abnormal cells
8
  • 2. expression screening
  • develop a cell-based functional assay for the
    abnormality
  • (e.g., a transport assay)
  • construct cDNA library in a way that will allow
    expression of
  • protein in mammalian cells
  • inject groups of cDNA clones into cells and
    assay function
  • narrow down cDNA clones using smaller groups of
    clones until
  • the function is observed with a single cDNA
    species
  • inject groups of cDNA clones
  • if the function being assayed is
  • observed, divide the group of clones
  • into smaller groups and retest
  • continue process of testing smaller
  • groups until the function being
  • assayed is obtained with one clone
  • for example, inject clones
  • and test cells for transport activity

left arm
right arm
human cDNA insert
mammalian promoter
9
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