एक ऐल्लि विशिष्ट जीन एक्सप्रेशन आनुवंशिक संघों के कार्यात्मक आधार टेस्ट परख
Summary
आनुवंशिक संघों अक्सर एक कार्यात्मक स्तर पर अस्पष्टीकृत रहते हैं. इस विधि के लिए जीन अभिव्यक्ति पर लिखित SNPs के लिए विषमयुग्मजी कोशिकाओं का विश्लेषण करके phenotype – जुड़े आनुवंशिक मार्करों के प्रभाव का आकलन करना है. प्रौद्योगिकी MALDI-TOF मास स्पेक्ट्रोमेट्री द्वारा सटीक माप एलील विशिष्ट प्राइमर विस्तार उत्पादों यों की अनुमति देता है.
Abstract
The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer’s specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.
Protocol
Discussion
इस विधि प्रतिलेख स्तर में vivo एलील विशेष अंतर का आकलन करके रोग से जुड़े जीन की अभिव्यक्ति पर डीएनए वेरिएंट के प्रभाव के मूल्यांकन के लिए सक्षम बनाता है. इस प्रोटोकॉल में रिश्तेदार प्रतिलेख बहुतायत MALDI-TOF लेकिन अन्य एलील विशिष्ट ऐसे 6,7 TaqMan के रूप में मात्रा का ठहराव, की अनुमति प्रौद्योगिकियों के द्वारा मापा जाता है, इस्तेमाल किया जा सकता है. इस दृष्टिकोण के प्रमुख सीमा कोडन लिखित मार्करों की उपलब्धता है. सिद्धांत पर आधारित तरीके यहाँ वर्णित है, लेकिन बहुरूपताओं के विभिन्न वर्गों के लाभ लेने, वर्णित किया गया है. haploChip परख एलील विशेष transcriptional शुरू करने या एक जीन जो chromatin immunoprecipitated द्वितीय 8 पोलीमरेज़ आरएनए विशिष्ट एंटीबॉडी के साथ अलग सामग्री में मौजूद होगा के अंत साइट के 1 केबी के भीतर स्थित मार्कर का उपयोग कर अभिव्यक्ति के उपाय. वैकल्पिक रूप से, intronic SNPs जब टेम्पलेट heteronuclear 9 शाही सेना इस्तेमाल किया जा सकता है .
विशिष्ट प्रोटोकॉल यहाँ वर्णित है, haploChip परख के साथ संयोजन में सफलतापूर्वक इस्तेमाल किया गया है डिस्लेक्सिया (या पढ़ने विकलांगता) के लिए एक उम्मीदवार जीन की पहचान. शुरू में एक आनुवंशिक संघ अध्ययन एक डीएनए डिस्लेक्सिया और जो तीन 10 जीनों फैले था के साथ जुड़े अनुक्रम की पहचान की . एलील विशिष्ट जीन की अभिव्यक्ति परख से पता चला है कि डिस्लेक्सिया जुड़े डीएनए वेरिएंट अभिव्यक्ति भिन्नता की उपस्थिति में केवल तीन जीनों के एक लिए मनाया था, लेकिन दो अन्य नहीं 11.
Disclosures
The authors have nothing to disclose.
Acknowledgements
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| PBS | Sigma | P-4417-100TAB | ||
| SDS | Biorad | 161-0416 | ||
| NaCl | VWR | 27788.366 | ||
| Tris | Sigma | T1503-1KG | ||
| EDTA | Sigma | E5134-500G | ||
| RNase A | Qiagen | 19101 | ||
| Proteinase K | Sigma | P2308-500MG | ||
| Phenol: chloroform | Sigma | 77617 | ||
| Chloroform | VWR | 100777C | ||
| Ethanol | Sigma | 32221-2.5L | ||
| Trizol | Invitrogen | 15596-018 | ||
| RNeasy kit | Qiagen | 74104 | ||
| SuperScript III Reverse Transcriptase | Invitrogen | 18080-044 | ||
| Immolase Taq | Bioline | BIO-21048 | ||
| dNTP | Sigma | DNTP100A | ||
| Exonuclease 1 | NEB | M0293S | ||
| Shrimp Alkaline Phosphatase | GE Healthcare | E70092Z | ||
| SpectroCLEAN resin | Sequenom | 10053 | ||
| MassEXTEND ddNTP/dNTP mix | Sequenom | Varies according to assay design | ||
| MassEXTEND thermosequenase | Sequenom | 10052 | ||
| Equipment | ||||
| Chilled microcentrifuge | Eppendorf | |||
| NanoDrop Spectrophotometer | Thermo Scientific | |||
| SpectroPOINT nanolitre dispenser | Sequenom | |||
| SpectroREADER mass spectrometer | Sequenom |
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