Feb 22, 2019 - 01:27 PM
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The DYS464X (Extended) Test

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Definition of DYS464X

DYS464X is a special method of testing the DYS464 STR alleles. R1b males have a G/C SNP in some of their DYS464 flanking regions very close to the reverse primer of the DYS464 locus. The DYS464X test uses altered primer sequences that end with their last base exactly at the SNP. One primer ends with a "G", while the other primer ends with a "C". These primers allow us to test the different SNP variants and their corresponding DYS464 STR alleles separately.

If the primer ending with G yields a peak, the allele is classed as a G-type.

If the primer ending with C yields a peak, the allele is classed as a C-type.

DYS464-forwardFAM-ttacgagctttgggctatg (exactly from the Redd paper)
DYS464-backward-G-Type JOE-cctgggtaacagagagactctttcag (ending with G)
DYS464-backward-C-Type TMR-cctgggtaacagagagactctttcac (ending with C)

PCR parameters: 30 x (90, 65, 70) and 60 end-elongation 1/2 h

Peaks with a green fluorescence (JOE) are called G type.
Peaks with a yellow fluorescence (TMR) are called C type.

There are some sample electropherograms in my library:

If you want to see a sequence alignment of the DYS464 DNA sequences and the primers you can download a BioEdit file from my alignment library:
[DYS464 Sequence Alignment]
The BioEdit alignment editor for Windows software can be downloaded from here:

What is the use of the DYS464X test?

Most R1b males that we have tested show 3 C-types and 1 G-type.
All other haplogroups (including R1a) show 4 G-types.

1.)The DYS464X method can verify, if a person is really R1b
2.)In many cases we can determine, if a single large peak really represents two different alleles
3.)Some DYS464 patterns look similar when the conventional test is used, but really consist of completely different alleles. For example:
14c-15c-17c-17g is completely different from
but this wouldn't be seen with the conventional typing method.
4.)DYS464X is a method that helps us observe recombinational loss of heterozygosity (recLOH)

Some DYS464 examples from an arbitary surname project:
(conventional results)

surname A, 1: 15-15-16-17
surname A, 2: 15-15-16-17
surname A, 3: 15-15-16-17
surname B, 1: 15-15-16-18
surname C, 1: 15-15-15-17

If all the 5 persons are R1b then they can be typed with an extended DYS464 test.

We might get the following results:

surname A, 1: 15c-15c-16g-17c
surname A, 2: 15c-15c-16g-17c
surname A, 3: 15c-15c-16c-17g
surname B, 1: 15c-15c-16g-18c
surname C, 1: 15c-15c-15g-17c

From that result we could say:

- A1 and A2 match completely
- A3 has at least a genetic distance of 2 from (A1 & A2)
- B1 has a genetic distance of 1 from (A1 & A2)
- C1 has a genetic distance of 1 from (A1 & A2)

I hope this is not too technical! Feel free to ask questions.

Thomas Krahn


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