From m.shariflou usyd.edu.au Thu Apr 5 08:36:46 2007
Subject: Conclusion on DNA extraction protocols/kits for SNP
genotyping
Date: Thu, 5 Apr 2007 16:41:05 +1000
From: "Mohammad Shariflou" <m.shariflouusyd.edu.au>
To: Multiple Recipients of <angenmapanimalgenome.org>
Dear AnGenMappers
As you remember I had the following question.
> Is there any generic protocol or DNA extraction kit, so that the
> resulting DNA can be used for genotyping platforms offered by different
> service providers?
I have received overwhelming responses, which is great to see such an
active community we have. I received 13 responses from which few of them
only expressed their interest to here the answer.
My conclusion is that we need a high quality DNA with accurate measurements
of DNA concentrations. The general consensus is that we can use a standard
protocol, and I did not receive any specific recommendation on a particular
kit.
As for appreciation of the correspondents, and also to provide you a
complete reference, I have summarized all the answers as you would see
after my message.
I greatly appreciate your contributions.
Regards
Mohammad
==================================
Dr Mohammad Reza Shariflou
Senior Researcher, SheepGenomics
Reprogen, Faculty of Veterinary Sciences
Level 5, Room 513, Gunn Bld, B19
University of Sydney, NSW 2006, Australia
Email: mohammadvetsci.usyd.edu.au
Tel: 612 9351 4789
Fax: 612 9351 3957
==================================
... my lab has done a substantial amount of the extractions for that
project. The extracted samples have been used on the Affymetrix,
Illumina and ABI (SNPlex) platforms. The key is that for Illumina and
ABI the concentration must be determined by PicoGreen assay because
those assays are sensitive to the presence of ssDNA. We use a basic
proteinase K extraction from semen, wbc or muscle samples Clare A. Gill,
clare-gilltamu.edu).
...I had to extract 1800 DNAs from chicken blood which was tored for
over more than 5 years. I used the commercial kit from PUREGENE. This
works fine, is easy, fast and cost-effective. From 25 µl of blood I
could isolate up to 20 µg. I will use the DNA for SNP genotyping with
the illumina Infinium II assay. Illumina does not recommend any special
kit. I do not think it is that important which kit you use. The thing is
you need pure, intact DNA (Pieter van, Pieter.vanAswur.nl).
...I have used the salt method (enclosed) for DNA extraction and used the
DNA for a range of projects. These days I run a human genetics lab and
we continue to use salt extraction of 10 ml blood as the main source of
DNA for SNP genotyping platforms including PCR-RFLP, TaqMan, Sequenom
and Illumina. There are a few critical points in the extraction process
and you need to standardise the protocols. I also think it is important
to carefully measure the DNA concentration and standardise the samples
before using high throughput genotyping platforms (Grant Montgomery,
Grant.Montgomeryqimr.edu.au).
...We use the standard protocol of salt and ethanol precipitation, from
about 300ml of blood collected in a blood bag. It was published about 1990
or so Montgomery and Sise from memory. DNA from this procedure has been
used for numerous tasks including extensive resequencing undertaken as part
of the hapmap project and the success rate has been very high.We have had
no problem with what we have used it for either.(john McEwan,
john.mcewanagresearch.co.nz).
...we use a simple salt out procedure. The DNA is used mainly for multiplex
microsatellite analyses and should be suitable for most SNP-analyses
protocols. The nice thing is that you do not need expensive kits and the
chemicals are easily to prepare and store [A detailed technical protocol
supplied] (Johannes Buitkamp, johannes.buitkamplfl.bayern.de).
...We are using Wizard genomic DNA purification system by using 96 well
format and works pretty well, as well as it is cheaper that the one from
QIAGEN. The yield is about 50-100 ng in 100 ul. We are using at the
moment microsatellites, AFLP and RAPD, but we will soon start with SNPs
in the 3130 Applied Biosystems (Marta Hern ndez,
ita-herpermaitacyl.es).
...We have used the Promega Wizard kit in 96-well format to extract DNA
from pig tails or other tissues. we use it routinely for genotyping on
the Sequenom MassArray system. We've used it for the hME and iPLEX
chemistry, which uses 2.5 - 10 ng per 5 ul reaction in 384-well format.
These are multiplex reactions; I have done up to 20 primer pairs and we
should be able to multiplex up to 30. We have also used it for PCR
sequencing and microsatellites (Dan Nonneman,
dan.nonnemanars.usda.gov).
...I am a final year PhD student and I have done exactly what you want to
do. You are right in saying that the kits only provide a small amount of
DNA and also for extraction of DNA from sheep blood they generally don't
provide DNA of sufficient quality for PCR. I established a "DNA bank"
for the sheep breed I am working on, and I would recommend that you do
the same. I have a protocol for the extraction DNA from sheep blood that
gives quantities in the region of several mg of DNA. This procedure also
provides a long term storage of the DNA. I have tried other procedures
and none work as well as this. Details of protocol supplied (Dawn
Howard, Dawn.Howardteagasc.ie).
... We extract DNA from cow blood and milk samples. Extraction from
blood is easy and yields high quantity and quality of DNA, that we use
for genotyping using tetra primer ARMS PCR procedure. we use a standard
phenol chloroform extraction procedure as described by winfrey rott
wortman in his book unraveling DNA (Molecular Biology for the
Laboratory). ... I think this should work for you. You might want to
confirm if phenol chloroform extraction works with your SNP platform. As
far as SNP chip is concerned, the quality of DNA matters. As long as you
have a good quality of DNA, it should be working on a SNP chip. But you
can always confirm that from whereever you are getting the SNP chips
from (Sameer Pant spantuoguelph.ca).
...I have a protocol that although is not very fast, not very expensive. It
is by the of eritrocites lisis technique, with sterile water, proteinaza
K, and RNA'asa, in the lisis buffer. The amount can be from 1 mililiter
to 10 mililiter (Cervantes Acosta (mailto:pcervantesuv.mx).
|
