Sequence-Specific "Gene Signatures" can be Obtained by PCR with Single Specific Primers at Low Stringency
| Title: | Sequence-Specific "Gene Signatures" can be Obtained by PCR with Single Specific Primers at Low Stringency |
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| Authors: | Pena, Sergio D.J.; Barreto, Guillermo; Vago, Annamaria R.; De Marco, Luiz; Reinach, Fernando C.; Neto, Emmanuel Dias; Simpson, Andrew J.G. |
| Publisher: | Proceedings of the National Academy of Sciences of the United States of America |
| Date Published: | March 01, 1994 |
| Reference Number: | 110 |
Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)
For example, on one of their test cases, the authors examined a family with X-linked nephrogenic diabetes insipidus (XNDI). XNDI involves a mutation of the vasopressin-2 receptor (AVPR2) gene. The authors amplified a fragment of one of the parts of the NDI patient's AVPR2 gene that contain part of the code for the vasopressin-2 receptor. They knew that this segment contained the mutations which caused the XNDI.
This segment, along with the corresponding AVPR2 segment from both the mother and the father of the NDI patient, all underwent a basic amplifying procedure called polymerase chain reaction (PCR). These segments were then subjected to LSSP-PCR. This procedure showed that the father's AVPR2 gene segment had a completely different "signature" or identifying pattern than did his son's gene segment. The son had XNDI. The signatures were clearly different even though the mutation in the segment that resulted in NDI consisted of just a single-base change (bases are the building blocks of the gene) that resulted in the substitution of a single amino acid substitution in the vasopressin-2 receptor the AVPR2 gene helped produce.
The fact that the LSSP-PCR technique shows distinct genetic signatures even when the mutation is a single base chain, and the fact it can do so relatively quickly and effectively, makes this procedure very useful for researchers.