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microarrays
What is a DNA Microarray?
DNA Microarrays are solid supports onto which the sequences (-) from different genes are attached.
The supports themselves are often microscope slides. They could also be silicon chips or nylon membranes.
The DNA is printed, spotted, or even synthesized directly onto the support (PCR).
To array means "to place in an orderly arrangement".
The gene sequences in a microarray are attached to the support in an orderly way, because we use
the location of a spot to identify a gene sequence.
The spots are either DNA, cDNA, or oligonucleotides.
---------------------------an array: colección, selección.
support: apoyo, ayuda, respaldo
microscopic slides: placas de microscopio.
spot: un lugar en que varios ejemplares de la secuencia de un gen son atadas.
a chip is small broken or cut off piece, as of wood, stone, or glass.
biochip is a microchip that uses tiny strands of ssDNA to latch onto and quickly recognize
thousands of genes at a time; intended for use in a biological environment
to latch onto sb (colloquial) vt: pegarse a alguien coloquial
Let's take the Affymetrix chips as an example:
preparation of a GeneChips (Affymetrix: http://www.affymetrix.com/index.affx)
Masks
To start the process, a quartz wafer is coated with a light-sensitive compound (mask).
This mask prevents coupling between the wafer and an unwanted nucleotide.
The surface is then covered with a solution containing either adenine, or thymine, or cytosine,
or guanine.
Coupling of the nucleotid to the glass occurs in those regions that have been deprotected
through illumination.
for example:
The light is used to deprotect sites where A’s are to be added.
The wafer is then flooded with a solution contianig A’s. The A’s attached only where
the wafer is deprotected. But the A’s that were added have also a protection which prevent
other nucleotides to attach.
The laser light deprotect now places where G’s should be attached.
A solution containing G’s is added....
Then deprotection where T’s should be attached …
The process is stopped when the attached oligos are ± 25 nt long.
Preparation of probes:
1. Preparation of poly A mRNAs of the sample we want to test.
2. Synthesis of single-stranded cADNs (ss cADN), using a primer made of
poli-dT connected to the T7 promoter sequence (blue rectangle below) , poly A mRNAs. and
RT DNA pol
3. Synthesis of double-stranded cADNs (ds cDNAs) containing the T7 promoter sequence.
4.
5.
6.
These cADNs serve as templates for in the "in vitro" transcription of complementary ARN
(cARN) by T7 RNA polymerase.
These cARN are biotin labeled.
They are fragmented. The fragments are be used as probes to check the arrays.
The chips is the introduced in an expensive machine…
Mismatch spots:
Each spot is designed to find whether or not a complementary sequence of (RNA or DNA)
is present in the sample.
Several spots are made for the measurement of each RNA or gDNA.
The length of the spot (25-mer) confers a high specificity.
To increase the sensitivity and reproducibility, multiple spotss are used.
22 spots are routinely used for each RNA measurement and 40 for each gDNA.
For each spot that matches its target sequence, there is a mismatch spot.
A mismatch spot contains a single mismatch located directly in the middle
of the 25-base sequence.
The perfect spote provides measurable fluorescence when sample binds to it.
the other spot is used to detect any contaminating fluorescence within that measurement.
chromatin precipitation
When a protein mixed with a DNA sequence slows down (shift) the migation of that DNA sequence
it proves that this protein is able to bind this DNA sequence “in vitro”. When one mix the same
protein, its specific antibody and the same DNA sequence, the migration of the DNA sequence
is more slowed down (supershift)…
But the condition is artificial in the sense that this protein “in vivo” could never meet the DNA
sequence.
If this protein binds DNA in real life, we should use chromatin precipitation.
First we sonicate the chromatin in a method standardized to give for example DNA sequences of
± 2000 bp. the we add the antibody to the molecule we suppose to be bound. By centrifugation we
precipitate the antigen antibody complex. This should precipitate the sequence of DNA to which
the protein is bound:
We extract the DNA from the precipitate. If the protein binds really to DNA,
we can, using primers, amplify the sequence at which the protein is supposed to
bind.
The shift is used to select binding sequences
chromatin precipitation is used to verify if the protein really binds.
screening
Megaprime is a labelling method for DNA fragments. It uses random sequence hexanucleotides
to prime DNA synthesis on denatured DNA template, at numerous sites along it length.
The primer-template complex is a substrate for the Klenow fragment of polymerase I.
By substituting a labelled nucleotide for a non labelled one the synthetized fragments,
these radioactive labelled fragments can then be used as sensitive hybridization probe for a wide
range of filter based applications
labelling
Megaprime is a labelling method for DNA fragments. It uses random sequence hexanucleotides
to prime DNA synthesis on denatured DNA template, at numerous sites along it length.
The primer-template complex is a substrate for the Klenow fragment of polymerase I.
By substituting a labelled nucleotide for a non labelled one the synthetized fragments,
these radioactive labelled fragments can then be used as sensitive hybridization probe for a wide
range of filter based applications
labelling using a phage SP6 or T7 RNA pol and labelled nucleotides
phage and cosmid screening
A phage or cosmid dilution is made to get 20.000 to 50.0000 pfu or cfu.
pfu= plaque forming unit
cfu= colony forming unit
phages
cosmids
PACs
BACs
YACs
Shotgun sequencing
'Shotgun sequencing''' is a method used in genetics for sequencing long DNA strands.
Since the chain termination method can only be used for fairly short strands (400 nt), it is necessary
to divide longer sequences up and then "assemble" the results to give the overall sequence.
Chromosome Walking
'Chromosome walking''' is a method in genetics for identifying and sequencing long parts of a DNA strand,
e.g., a chromosome. As the traditional chain termination method does not allow long DNA strands to be
sequenced, this method works by dividing the long sequence into several consecutive short ones.
The basic technique is as follows: A primer that matches the beginning of the DNA to sequence is used
to synthesize a short DNA strand complementary to the unknown sequence, starting with the primer
(see PCR). The new short DNA strand is sequenced using the chain termination method.
The end of the sequenced strand is used as a primer for the next part of the long DNA sequence.
That way, the short part of the long DNA that is sequenced keeps "walking" along the sequence.
The method can be used to sequence entire chromosomes (thus, chromosome walking)
Chromosome jumping.
Chromosome jumping:
A research technique developed to move rapidly up or down a chromosome in search of a particular gene.
It enables researchers to make large jumps over uninformative regions of DNA.
Chromosome jumping is used to bypass regions difficult to clone, such as those containing
repetitive DNA, that cannot be easily mapped by chromosome walking, and is useful in moving along
a chromosome rapidly in search of a particular gene.
In chromosome jumping, the DNA of interest is identified, cut into fragments with restriction enzymes,
and circularised (the beginning and end of each fragment is joined together to form a circular loop).
From a known sequence a primer is designed to sequence across the circularised junction.
This primer is used to jump 100 kb-300 kb intervals: a sequence 100 kb away would have come near
the known sequence on circularisation. Thus, sequences not reachable by chromosome walking
can be sequenced. Chromosome walking can be used from the new jump position (in either direction)
to look for gene-like sequences, or additional jumps can be used to progress further along the chromosome
http://www.hhmi.org/genetictrail/a120.html
Southern (DNA) and Northern (RNA) analysis
Southern analysis:
Typically you prepare genomic DNA.
You cut it into small fragments, usinq a retriction enzyme.
The hydrolysate is submitted to agarose gel electrophoresis.
The gel is put into NaOH to denaturate the DNA it contains.
It is maintained denatured during the capillary transfer by the NaCl 3M.
The capillary transfer aims to transfer the DNA from the gel to a filter.
Once the transfer is done, the DNA is covalently attached to the membrane by a 3 min UV irradiation.
The filter is put into a small freeze bag.
The hybridation solution and the probe are added
Bubbles are expelled and the bag is hermtically closed.
During 16 hours (one night), the liquid phase ils allowed to run over the solid phase.
An autoradiography is done (one or two day exposition).
The autoradiography is revealed.