Simultaneous quantitative and allele-specific expression analysis with real competitive PCR

BMC Genetics

Charles R Cantor³  Andreas Braun³  Adelbert A Roscher²  Esther Maier²  Chunming Ding¹ 

[1] Bioinformatics Program and Center for Advanced Biotechnology, Boston University, Boston, MA 02215 USA;Children's Hospital, University of Munich, Lindwurmstrasse 4, 80337 Munich, Germany;SEQUENOM, Inc., San Diego, CA 92121 USA

关键词: heterozygous;    PCR;    MALDI-TOF mass spectrometry;    gene expression;    allele-specific;   

Others  :  1143843
DOI  :  10.1186/1471-2156-5-8

 received in 2004-02-20, accepted in 2004-05-05,  发布年份 2004

【 摘 要 】

Background

For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis.

Results

A transcribed mutation such as a single nucleotide polymorphism (SNP) is used as the marker for allele-specific expression analysis. A synthetic mutation created in the competitor is close to a natural mutation site in the cDNA sequence. PCR is used to amplify the two cDNA sequences from the two alleles and the competitor. A base extension reaction with a mixture of ddNTPs/dNTP is used to generate three oligonucleotides for the two cDNAs and the competitor. The three products are identified and their ratios are calculated based on their peak areas in the MALDI-TOF mass spectrum. Several examples are given to illustrate how allele-specific gene expression can be applied in different biological studies.

Conclusions

This technique can quantify the absolute expression level of each individual allele of a gene with high precision and throughput.

【 授权许可】

   
2004 Ding et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Cowles CR, Joel NH, Altshuler D, Lander ES: Detection of regulatory variation in mouse genes. Nat Genet 2002, 32:432-437.
• [2]Momparler RL: Cancer epigenetics. Oncogene 2003, 22:6479-6483.
• [3]Lo HS, Wang Z, Hu Y, Yang HH, Gere S, Buetow KH, Lee MP: Allelic variation in gene expression is common in the human genome. Genome Res 2003, 13:1855-1862.
• [4]Bray NJ, Buckland PR, Owen MJ, O'Donovan MC: Cis-acting variation in the expression of a high proportion of genes in human brain. Hum Genet 2003, 113:149-153.
• [5]Yan H, Yuan W, Velculescu VE, Vogelstein B, Kinzler KW: Allelic variation in human gene expression. Science 2002, 297:1143.
• [6]Butz JA, Yan H, Mikkilineni V, Edwards JS: Detection of allelic variations of human gene expression by polymerase colonies. BMC Genet 2004., 5(3)
• [7]Growdon WB, Cheung BS, Hyman BT, Rebeck GW: Lack of allelic imbalance in APOE epsilon3/4 brain mRNA expression in Alzheimer's disease. Neurosci Lett 1999, 272:83-86.
• [8]Lambert JC, Perez-Tur J, Dupire MJ, Galasko D, Mann D, Amouyel P, Hardy J, Delacourte A, Chartier-Harlin MC: Distortion of allelic expression of apolipoprotein E in Alzheimer's disease. Hum Mol Genet 1997, 6:2151-2154.
• [9]Ding C, Cantor CR: A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. Proc Natl Acad Sci U S A 2003, 100:3059-3064.
• [10]Gardner TS, di Bernardo D, Lorenz D, Collins JJ: Inferring genetic networks and identifying compound mode of action via expression profiling. Science 2003, 301:102-105.
• [11]Maier EM, Kammerer S, Muntau AC, Wichers M, Braun A, Roscher AA: Symptoms in carriers of adrenoleukodystrophy relate to skewed X inactivation. Ann Neurol 2002, 52:683-688.