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
-

-
-
-
-
-


-
-
104
102
-
-
-
-
-