Advantages of RT-PCR and denaturing gradient gel electrophoresis for analysis of genomic imprinting: Detection of new mouse and human expressed polymorphisms
✍ Scribed by Mitsuyoshi Nakao; James S. Sutcliffe; Arthur L. Beaudet
- Book ID
- 101263785
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 489 KB
- Volume
- 7
- Category
- Article
- ISSN
- 1059-7794
No coin nor oath required. For personal study only.
✦ Synopsis
Communicated by lohn A. Phillips 111 Genomic imprinting, or differential expression of alleles based on parental origin, is documented for numerous mouse and human loci and is implicated in various phenotypes such as Wilms tumor, Beckwith-Wiedemann syndrome, Prader-Willi syndrome, and Angelman syndrome. Improved methods would facilitate the analysis of imprinting, and we describe a simple strategy designed to analyze transcripts for imprinting in mouse and human using reverse transcription-polymerase chain reaction (RT-PCR) in combination with GC-clamped denaturing gradient gel electrophoresis (DGGE). As a demonstration, novel polymorphisms in the untranslated portions of mRNA between CBA/Nj and Skive strains of mice were identified and used to document paternal expression of small nuclear ribonucleoprotein associated polypeptide N (Snrjm) in brain, maternal expression of HI9 in liver, and biallelic expression of glyceraldehyde 3-phosphate dehydrogenease (Gapd) in liver. The method was also used to demonstrate a new polymorphism and monoallelic expression of HI9 in human peripheral leukocytes. Assessment of imprinting for novel or unstudied transcripts requires identification and analysis of polymorphisms at the RNA level, and we believe that RT-PCR with DGGE is a preferred method for this application, with advantages over nuclease protection and other methods.