Title: a' Library screening: plaque hybridization
110. 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
-
-
-
-
-