Human DNA fingerprinting - (Dec/14/2010 )

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HomeBrew on Fri Dec 24 22:02:31 2010 said:

Ok, I get it now. Depending on the magnitude of the changes between these alleles, you might be able to separate them with agarose in a lithium borate buffer system (see Brody, J.R., Calhoun, E.S., Gallmeier, E., Creavalle, T.D., Kern, S.E. (2004): Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques 37(4):598-602.), or sodium borate system (see Brody, J.R., Kern, S.E. (2004): Sodium boric acid: a tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36(2):214-215.), both of which have superiour resolving power compared to TAE- or TBE-based gels. Not as powerful as polyacrylamide, but easier to work with...

Thank you for your response. I have heard of these techniques before but am yet to try them, although the technique I am already using works in the sense that the bands are visible.

The agarose gel size I have been using so far is approx. 120mm x 90mm. Do you think this is too small a gel to separate the gel bands to see differentiation between individuals profiles?...since the 6 bands that I am seeing for each individual's DNA profile are in the same place as each other, so there is no differentiation between them.

-Baileys-

Baileys on Sun Dec 26 20:13:37 2010 said:

The agarose gel size I have been using so far is approx. 120mm x 90mm. Do you think this is too small a gel to separate the gel bands to see differentiation between individuals profiles?

Again, it depends on the magnitude of differeces you expect to see in such an analysis, something I still don't have clue on. How big are the STR's? Do you expect a 3-bp difference between fragments, a 30-bp difference, what?

-HomeBrew-

HomeBrew on Sun Dec 26 23:13:20 2010 said:

Baileys on Sun Dec 26 20:13:37 2010 said:

The agarose gel size I have been using so far is approx. 120mm x 90mm. Do you think this is too small a gel to separate the gel bands to see differentiation between individuals profiles?

The STR's are 4 bp in size
I don't know exactly what bp difference I will see between fragments, but a minimum of 4 bp.

-Baileys-

Hi Baileys,

I do STR fingerprinting but never used agarose. Why don't you label your primers and run them on a 3100 like everyone?

-Maddie-

Maddie on Wed Dec 29 18:55:01 2010 said:

Hi Baileys,

I do STR fingerprinting but never used agarose. Why don't you label your primers and run them on a 3100 like everyone?

One more thing. It is entirely possible to get similar profiles with only 3 loci. Most kits use 17. This is especially true if you have the most frequent alleles. For example, with D18, alleles 14, 15, 16 and 17 are much more frequent than the others. See http://spsmart.cesga.es/popstr.php?dataSet=strs_local

What are the other 2 loci you are targeting?

-Maddie-

Maddie on Wed Dec 29 19:09:39 2010 said:

Maddie on Wed Dec 29 18:55:01 2010 said:

Hi Baileys,

I do STR fingerprinting but never used agarose. Why don't you label your primers and run them on a 3100 like everyone?

Hi Maddie,

I'm afriad I haven't heard of a "3100" before. The whole STR analysis of DNA is very new to me at the moment.
Additionally, the three loci I am looking at are D18S51, TH01 and D13S317.

-Baileys-

Baileys,

I meant a sequencer (genetic analyzer) from ABI (Applied Biosystems). They have 310, 3100, 3130, 3730 amd now 3500.
https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601914

In crime lab, scientists use kits to do fingerprints. ABI sells Identifiler and MiniFiler (for inhibited and/or degraded samples)
https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601705
https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=603805

Promega sells Poweplex 16
http://www.promega.com/applications/hmnid/productprofiles/pp16/Default.htm

On top of giving STR profiles, these kits allow you to determine the sex of the individual. They all contain a master mix (buffer etc..) and a primer mix. You can have up to 17 pairs of primers that have dyes attached which allows your PCR product to be visualized on the sequencer. The kits also contain an allelic ladder (a mix of all the alleles from all the loci analyzed). This is a very straight forward process: you mix the master mix, the primer mix, add DNA, water and Taq (unless it's MiniFiler where the Taq is already in the mix) then PCR and after that you can load the PCR product on the sequencer with no clean-up step. It is quite fast and very easy if you can afford the kit.

If you are a beginer, I would adivse you to go to John Butler's web site
http://www.cstl.nist.gov/biotech/strbase/

This is very complete. You can also get one of his books
http://www.amazon.com/Fundamentals-Forensic-Typing-John-Butler/dp/0123749999/ref=sr_1_2?ie=UTF8&s=books&qid=1293735347&sr=1-2
or
http://www.amazon.com/Forensic-DNA-Typing-Second-Technology/dp/0121479528/ref=sr_1_1?ie=UTF8&s=books&qid=1293735347&sr=1-1

If you cannot buy the kits, you can of course do your own multiplex with labeled primers but you will need a sequencer. I really don't think agarose gels are precise enough.
Let me know if you have more questions.

-Maddie-

I was really hoping someone who knew something about this (i.e. not me) would come along and give Baileys some advice. Thanks, Maddie!

-HomeBrew-

Maddie on Thu Dec 30 18:59:19 2010 said:

Thank you very much for this information and the links you have given. They are very useful. I'll certianly be looking at John Butler's website

-Baileys-

You are very welcome gentlemen

HAPPY NEW YEAR !!!! May 2011 be full of progress.

-Maddie-

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