19 replies to this topic

[Baileys]

I am making DNA fingerprints using human DNA from 6 unrelated individuals. I use chelex extraction, PCR and agarose gel electrophoresis. I am looking at 3 loci, and get 6 bands seperating on the gel for each person. However, these 6 bands are in the same place, so I cannot differentiate between each person because it looks as if they all have the same DNA fingerprints. I know that sometimes bands will appear in the same place between individuals but I think it is unusual that this will be the case for 3 loci from 6 different people. There should be at least some variation there. Does anyone know why this might be happening or possible soltuions to the problem?

[seanspotatobusiness]

I don't know squat about DNA fingerprinting but maybe it depends on the loci? Some are going to be more conserved than others, and you're looking for differences in sequence length? That's going to be a variation I'd expect to occur only in certain places, at much lesser frequency than, say, an SNP.

[Baileys]

I wondered if it was something to do with the loci too.

However, I am considering trying a different gel electrophoresis technique...perhaps a vertical gel, which should give better seperation than a small agarose gel so with any luck, the bands will be seperated with respect to each other.

I am using a DNA ladder in order to see what size the fragments are.

[HomeBrew]

Are you sure that agarose gel electrophoresis is the way to go (I also don't know anything about DNA fingerprinting)?  You will only be able to detect changes due to insertions or deletions of sufficient size to be resolved on a gel -- and the resolving power of standard agarose gel electrophoresis is not that great.  You'll never detect SNPs, which are much more likely than relatively large insertions or deletions.

What does the literature say?  Can you sequence PCR products from these loci?

[Baileys]

HomeBrew, on 23 December 2010 - 02:52 AM, said:

Are you sure that agarose gel electrophoresis is the way to go (I also don't know anything about DNA fingerprinting)?  You will only be able to detect changes due to insertions or deletions of sufficient size to be resolved on a gel -- and the resolving power of standard agarose gel electrophoresis is not that great.  You'll never detect SNPs, which are much more likely than relatively large insertions or deletions.

What does the literature say?  Can you sequence PCR products from these loci?

One of the loci I am looking at is called D18S51 and I have found a protocol for making a DNA fingerprint from just this one loci and they use the same procedures as me, except they are using blood as their sample. They also use 2% agarose gel, which is what I have been using, which for me has been unsuccessful in determining between people since all their bands lie next to each other in the same place. Do you think that a vertical polyacrylamide gel would be better for differentiation?

[HomeBrew]

Well, polyacrylamide has much greater resolving power -- it can resolve a one base pair difference, as we know from Sanger sequencing methods.  But no electrophoresis can differentiate fragments of the same length, so you will only see differences between individuals is there's been a deletion or insertion in the loci.  Is this common in this loci?

[Baileys]

HomeBrew, on 23 December 2010 - 06:12 PM, said:

Well, polyacrylamide has much greater resolving power -- it can resolve a one base pair difference, as we know from Sanger sequencing methods.  But no electrophoresis can differentiate fragments of the same length, so you will only see differences between individuals is there's been a deletion or insertion in the loci.  Is this common in this loci?

Thank you for your response.

I understand that electrophoresis can not differentiate fragments which are the same length...only frangments that are different lengths. The D18S51 locus is the locus that differentiates between people the most out of all of the loci used by the CODIS database. U.S Caucasians are more likelt to have either alleles 12, 13, 14, 15, 16 and 17 from this specific loci, whereas with U.S African-Americans, it is alleles 15, 16, 17, and 18.

Surely the different alleles of the three loci will have fragment bands in different places due to the difference in the number repeats of TCAT (or whatever the repeat sequence may be for each loci).

Edited by Baileys, 24 December 2010 - 07:21 AM.

[HomeBrew]

Baileys, on 24 December 2010 - 06:02 AM, said:

The D18S51 locus is the locus that differentiates between people the most out of all of the loci used by the CODIS database. U.S Caucasians are more likelt to have either alleles 12, 13, 14, 15, 16 and 17 from this specific loci is, whereas with U.S African-Americans, it is alleles 15, 16, 17, and 18.

I'm sure all that's true.  But what is the nature of the variation that defines these alleles?  Is it fragment length?

[Baileys]

HomeBrew, on 24 December 2010 - 07:16 AM, said:

Baileys, on 24 December 2010 - 06:02 AM, said:

The D18S51 locus is the locus that differentiates between people the most out of all of the loci used by the CODIS database. U.S Caucasians are more likelt to have either alleles 12, 13, 14, 15, 16 and 17 from this specific loci is, whereas with U.S African-Americans, it is alleles 15, 16, 17, and 18.

I'm sure all that's true.  But what is the nature of the variation that defines these alleles?  Is it fragment length?

Each short tandem repeat allele has a different length depending on the number of tandem repeats it contains e.g., the TH01 loci has a varying number of TACT repeats. So when the alleles are amplified by PCR, alleles of different lengths can be distinguished by gel electrophoresis.

[HomeBrew]

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...

[Baileys]

HomeBrew, on 24 December 2010 - 02:02 PM, 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.

[HomeBrew]

Baileys, on 26 December 2010 - 12:13 PM, 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?

[Baileys]

HomeBrew, on 26 December 2010 - 03:13 PM, said:

Baileys, on 26 December 2010 - 12:13 PM, 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?

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.

[Maddie]

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 29 December 2010 - 10:55 AM, 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...aSet=strs_local

What are the other 2 loci you are targeting?