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GSL - SNP Shot
SNP
GENOTYPING
SNP (Single Nucleotide
Polymorphism)
SNP stands for "single nucleotide
polymorphism". SNPs are the most common genetic variations and occur once every
100 to 300 bases. A key aspect of research in genetics is the association of
sequence variation with heritable phenotypes. It is expected that SNPs will
accelerate the identification of disease genes by allowing researchers to look
for associations between a disease and specific differences (SNPs) in a
population. This differs from the more typical approach of pedigree analysis
which tracks transmission of a disease through a family. It is much easier to
obtain DNA samples from a random set of individuals in a population than it is
to obtain them from every member of a family over several generations. Once
discovered, these polymorphisms can be used by additional laboratories, using
the sequence information around the polymorphism and the specific experimental
conditions.
SNP can be also used for study the allelic
variation in gene expression ("Science" v297, p1143, August 16, 2002 for mathods
please go to:
http://www.sciencemag.org/cgi/data/297/5584/1143/DC1/1).
AU GSL provides quick, low cost, and
high throughput SNP genotyping services
The actual cost can be as low as $0.45 per
sample and up to 10,000 samples can be analyzed per day.
Overview of the procedure
1. Template preparation (this step will be
conducted by researchers): target genomic DNA regions will be amplified by PCR,
the PCR products will be purified to remove dNTPs and primers.
2. Reaction preparation (this step can be
conducted by either AU GSL or by researchers): the purified PCR products (up to
10 different PCR products), primers (up to 10 different primers with different
sizes, at leaset 5 nt difference in length), and labeled ddNTPs and Taq
polymerase will be combined in a single tube.
3. Thermal cycling--Post-Extension
Treatment (this step can be conducted by either AU GSL or by researchers):
Perform thermal cycling, and then remove unincorporated ddNTPs with SAP or CIP.
4. GeneScan Analysis: The samples will be
run on ABI 3100 Genetic Analyzer and the data generated will be analyzied with
GeneScan software.
Detailed Protocols (from "ABI Prism
SNaPshot Multiplex Kit Protocol"):
1. Preparing Your PCR Template for
Primer Extension
After PCR amplification, the resulting
templates is in solution along with primers, dNTPs and enzyme and buffer
components. To avoid participation in the subsequent primer-extension reaction,
primers and unincoporated dNTPs must be removed. Applied Biosystems recommend
two methods to purify PCR products.
The first is to use PCR Clean up kits (P/N
1696513) from Roche Molecular Biochemicals to purify the PCR products. Refer to
the manufacture's instructions for the procedure.
The second is to use SAP and ExoI
treatment:
1) Add the following to the 15 ul of PCR
product:
5 units of SAP
2 units of Exo I
2) Mix throughly and incubate at 37C for 1
hour
3) Incubate at 75C for 15 min to
inactivate the enzymes
4) Keep on ice or at 4C, for longer
storage, store at -20C.
2. Preparing Your Sample Reactions
1) Mix equal volumes of PCR samples (up to
10) in one tube and place the tube on ice
2) Mix all the primers (up to 10) to be
used to give a final concentration of 0.2 uM for each primer. Place the primer
mixture on ice.
Note: the length of each primer should be
at least with 5 nt difference, e.g. 20mer, 25mer,30mer, 35 mer.....50mer, 55mer,
60mer etc.
3) Setting up sample reaction:
5 ul SNaPshot Multiplex Ready Reaction Mix
3 ul Pooled PCR products
1 ul Pooled primers
1 ul ddH2O
10 ul total reaction volume
3) Label two 0.2 ml tube, one for positive
control and one for the negative control.
add:
For Positive Control:
5 ul SNaPshot Multiplex Ready Reaction Mix
2 ul SNaPshot Multiplex Control PCR
products
1 ul SNaPshot Multiplex Control primer Mix
2 ul ddH2O
10 ul total reaction volume
For Negative Control:
5 ul SNaPshot Multiplex Ready Reaction Mix
0 ul SNaPshot Multiplex Control PCR
products
1 ul SNaPshot Multiplex Control primer Mix
4 ul ddH2O
10 ul total reaction volume
3. Thermal Cycling and Post-Extension
Treatment
PCR-Repeat following for 25 cycles:
96 C for 10 Sec
50 C for 5 Sec
60 C for 30 Sec
after 25 cycles,
4 C hold until ready for post extension
treatment
Post-Extension Treatment: (important, left
untreated, the unincorporated [F]ddNTPs will co-migrate with the fragmentsof
interest and interfere with the results):
1) Add one of the following to the
reaction mixture, mix thoroughly, and incubate at 37 C for 1 hour
* i) 1.0 unit of Shrimp Alkaline
Phosphatase (SAP) or
*ii) 1.0 unit of Calf Intestinal
Phospatase (CIP)
2) Deactivate the enzyme by incubating at
75 C for 15 min
3) Sample may be placed at 4C for up to 24
hours prior to electrophoresis on the 3100 systems, for storage longer than 24
hours, store the samoles at -20 C.
Sending your samples to run
electrophoresis on the ABI Prism 3100 Genetic Analyzer
SNP Order Form
I. If you performed SNaPshot reaction in
your own lab, send the final products to GSL at 33 Life Science Building for
electrophoresis on the ABI Prism 3100 Genetic Analyzer ($4.50/tube of your
sample).
II. For sending your samples to conduct
SNaPshot reaction in GSL ($8.00/well):
1) Pooling PCR amplified SNaPshot
templates: if you have multiple PCR amplified samples to run in a single well,
mix equal volums (e.g 2 ul each) of these products in a tube.
2) Pooling SNaPshot primers: all the
primers to be used in a single SNaPshot reaction should be premixed to give a
final concentration of 0.2 uM for each primer.
Fill out your
order form on line and send your samples to 33 Life Science Building (phone:
334-844-1619), Auburn University.
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