The human sex determining region on the Y chromosome has been identified and the gene has been termed as SRY. Mutations in the SRY gene have been found in XY females. Sex reversal in XY females results from the failure of the testis determination or differentiation pathways. Some XY females with gonadal dysgenesis have lost the SRY gene from the Y chromosome by terminal exchange between the sex chromosome or by other deletions or mutations affecting activity (1,2).

DNA analysis for a specific region of SRY together with alphoid repeat regions of the X and Y chromosome is used for accurate sex determination (in the absence of mutations involving SRY), and in the characterization of X-linked genetic diseases, Y chromosome anomalies such as XY females with gonadal dysgenesis, and for XO/XY mosaicism in patients with Turner syndrome. The DNA test involves the amplification of specific regions of X, Y and SRY. The presence of amplified product directly indicates the presence of the cognate DNA fragments on the chromosome. Normal XX females will amplify only X chromosome specific fragment showing double intensity as compared with amplification from normal XY male. SRY and Y fragments will only be amplified from individuals with a Y chromosome.

Material Supplied
Two lyophilized oligonucleotide primers are supplied. Please refer to item number on the top of this sheet (SRY, X or Y Genemer>ä ). Each tube contains 10 nmoles of the primer. The quantity supplied is sufficient for 400 regular 50>m l PCR reaction.

Protocol for SRY, X and Y DNA Genotyping
The following PCR* profile has been optimized for SRY, X and Y specific product amplification using the supplied Genemers>ä

A. Reconstitution
Stock solution: Add 50>m l sterile water to each tube containing the primer. The 10 nmoles of primer when dissolved in 50>m l water will give a solution of 200 >m Molar i.e. 200 pmoles/>m l.
Primer Mix: Prepare a 10 pmoles/>m l Primer Mix solution containing BOTH the primers. Example; add 180 >m l water to a new tube. To this tube add 10 >m l of EACH of the primer stock solution from #1 step above. Label this tube as Primer Mix 10 pmoles/>m l.

B. PCR* reaction (see Appendix for Details)

PCR Profile
Denaturation_______________94oC_____30 sec.
Annealingn_______55oC____30 sec.
Elongation________72oC____1 min.
30 cycles, 7 min. 72oC extension, 4oC soak.

C. Electrophoresis
Load samples to 1.5% agarose gel. Run at 90 mAmps for 2.5 hrs.

D. Results
-Normal female DNA should only amplify X specific fragment.
-Normal male DNA should amplify all fragments (SRY, X & Y)

Normal PCR amplified fragment size SRY______X______Y __________422 bp__130 bp_170 bp

Figure. SRY, X and Y PCR amplification gel profile. Lane 1, molecular weight marker. Lanes 2-4 male DNA, lanes 5-7 female DNA. Lanes 2 and 5 SRY amplification, lanes 3 and 6 X amplification, lanes 4 and 7 Y amplification. Note the absence of amplification of SRY and Y from female DNA (lanes 5 & 7).

 

References
1. Berta et al. (1990) Genetic evidence equating SRY and the testis-determining factor. Nature 348:448-451.
2. Jager et al. (1990) A human XY female with frame shift mutation in the candidate testis-determining gene SRY. Nature 348:452-453.
3. Witt, M. & Erickson, R.P. (1989) A rapid method for detection of Y-chromosome DNA from dried blood specimens by the polymerase chain reaction. Hum. Genet. 82:271-274.

Product

Size

Catalog No.

Price

Please inquire, Linkmers>ä

10nmoles

$75.00