Detecting Lesch-Nyhan syndrome by solid phase primer extension
Journal Article
·
· American Journal of Human Genetics
OSTI ID:134326
- Baylor College of Medicine, Houston, TX (United States)
A mutation detection method based upon the wild type human HPRT sequence is presented for identification of Lesch Nyhan syndrome. The technique consists of performing a biotinlyated PCR amplification of the region of interest, followed by isolation and purification of single stranded template using magnetic separation. Allele-specific primers are annealed adjacent to the potential mutation site on the template. A terminal fluorescent deoxynucleotide addition is performed with a DNA template-dependent polymerase to distinguish between the mutant and wild-type sequence. The products are purified from unincorporated ddNTPs, eluted and finally analyzed on an ABI 373 to identify the mutation. The length of an extension primer is used as a position signature for mutations. The fidelity of nucleotide incorporation provides an excellent signal-to-noise ratio for the detection of nine HPRT mutations within eight cell lines. This method should detect all types of mutations except for repeated sequences that are longer than the primers. Moreover, the method is being extended to a solid support assay, whereby the extension primers are attached to a two-dimensional glass surface. Following extension, the solid support is analyzed for radioactive incorporation. We have shown the sequence determination of a five base region of a wild-type sequence and two different HPRT mutations. As more dense oligonucleotide arrays are produced, this method could be extended to sequence the complete coding region of HPRT.
- OSTI ID:
- 134326
- Report Number(s):
- CONF-941009--
- Journal Information:
- American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
Similar Records
Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA
Single nucleotide primer extension to detect genetic diseases: Experimental application to hemophilia B (factor IX) and cystic fibrosis genes
DNA sequencing process using stable isotopes
Journal Article
·
Tue Feb 28 23:00:00 EST 1989
· Proceedings of the National Academy of Sciences of the United States of America; (USA)
·
OSTI ID:7018773
Single nucleotide primer extension to detect genetic diseases: Experimental application to hemophilia B (factor IX) and cystic fibrosis genes
Journal Article
·
Thu Feb 14 23:00:00 EST 1991
· Proceedings of the National Academy of Sciences of the United States of America; (United States)
·
OSTI ID:6107772
DNA sequencing process using stable isotopes
Patent
·
Mon Mar 25 23:00:00 EST 1991
·
OSTI ID:5018156