Advantages of RT-PCR and denaturing gradient gel electrophoresis for analysis of genomic imprinting: detection of new mouse and human expressed polymorphisms.
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 (Snrpn) in brain, maternal expression of H19 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 H19 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.
Nakao
M
M
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Sutcliffe
J S
JS
Beaudet
A L
AL
D000818:Animals; D001483:Base Sequence; D003433:Crosses, Genetic; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D018392:Genomic Imprinting / Q000235:genetics*; D006801:Humans; D007962:Leukocytes; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers