Subtractive Genomic Hybridization

Subtractive Genomic Hybridization
Subtractive hybridization is a powerful technique to study gene

expression in specific tissues or cell types or at a specific stage.
Cloned genes can sometimes by found by a negative approach.
Hybridization is used to remove genes shared by two organisms,
leaving behind only those that are unique.
Traditional procedures often are technically demanding and
labor-intensive methods that require large amounts of mRNA and
might give rise to falsely positive and unreproducible results.
Both labeled cDNA and cDNA/mRNA hybrids are immobilized.
The non-labeled mRNA molecules representing differentially
expressed genes are isolated
• Subtractive cDNA hybridization has been a powerful
approach to identify and isolate cDNAs of
differentially expressed genes.
• In general, they involve hybridization of cDNA from
one population (tester/ Subtractor) to excess of
mRNA (cDNA) from other population (driver) and
then separation of the unhybridized fraction (target)
from hybridized common sequences.
• The latter step is usually accomplished by
hydroxylapatite chromatography, avidin-biotin
binding , or oligo(dT)3o-latex beads
• 1. to find out the deleted DNA in one sample
To do subtractive hybridization, both the mutant and wild-type
DNA samples are cut into fragments of convenient size using a
restriction enzyme.
• Then the two sets of fragments are hybridized together.
• This will give hybrid molecules for all regions of the DNA except
the region of the deletion, which is present only in the wild-
type chromosome.
• If a large surplus of mutant DNA is used, all fragments of the
wild-type chromosome will be hybridized to mutant fragments
except the region corresponding to the deletion, which will be
left over.
• The single strands of this lone fragment will have to pair with
each other. Thus, we have subtracted out all the segments of
DNA that are not wanted.
2. To identify a set of genes that are expressed under
particular conditions
Method
1. Isolate the two mRNA pools of interest
• When working with RNA, always use RNase inhibitors to
prevent degradation of RNA
2. Generation of the biotinylated cDNA pool (subtractor
cDNA)
• The generation of biotinylated cDNA from mRNA is
accomplished by using 5’-biotinylated Oligo(dT) primer in
a Reverse Transcription reaction.
• Remove the mRNA from the mRNA/cDNA hybrids by
using RNase H. Inactivate RNase H prior hybridization by
phenol extraction
3. Hybridization
• Mix the target mRNA and the subtractor DNA in
about 100 µl of Hybridization Buffer.
• Hybridize at 68 °C overnight. To reduce the

stringency, you may lower the temperature to
42°C or even to room temperature.
• The salt concentration of the hybridization

buffer likewise affects the hybridization reaction:
the lower the salt concentration, the higher the
stringency.
Positional Gene Cloning
Positional cloning is a term derived from the late 1980s which basically was to be
contrasted to functional cloning, so probably we should define both. Functional
cloning was finding a gene by understanding something about what its function
is.
So the hemophilia gene was identified by knowing there was a problem with a
blood clotting factor and then figuring out what gene must have coded for that,
and isolating or cloning that gene.
But for most diseases, we don't have enough information to guess what the
function was, so positional cloning, which came into being as a need of trying to
identify the cause of things like cystic fibrosis, was a way of identifying the gene
by its position in the genome.
Basically, it's the position in the genome that you're trying to zero in on by a
series of steps that go from a larger view to narrower view to finally zeroing in on
the single base pair that's gone awry. And in many instances that's what you're
looking for.
• Positional cloning is the approach of choice for the
identification of genetic mutations underlying the
pathological development of diseases with simple
Mendelian inheritance.
• It consists of different consecutive steps, starting
with recruitment of patients and DNA collection,
that are critical to the overall process.
• A genetic analysis of the enrolled patients and
their families is performed, based on genetic
recombination frequencies generated by meiotic
cross-overs and on genome-wide molecular
studies, to define a critical DNA region of interest.

Subtractive Genomic Hybridization

Uploaded by

Copyright:

Available Formats

Subtractive Genomic Hybridization

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Subtractive Genomic Hybridization

Uploaded by

Copyright:

Available Formats

Subtractive Genomic Hybridization

Subtractive hybridization is a powerful technique to study gene

• Hybridize at 68 °C overnight. To reduce the

• The salt concentration of the hybridization

You might also like