Microassay for serial analysis of gene expression and applications thereof

摘要

The method that the tag library that method obtains can provide specific state biological sample,Including following consecutive steps:( 1 )Extraction single step mRNA uses oligomeric from a small amount of biological sample(Deoxythymidine)25 are covalently bound to paramagnetic beads( 2 )Generate double-stranded cDNA library,From the mRNA,It rives to gained cDNAs( 3 )Use Sau3A I,( 4 )Separate the cDNAs two halves equal parts split,( 5 )Ligaturing the described two aliquots for including in each of the cDNA has one of following formula GATCGTCCC-X1 or GATCGTCCC-X2 by a doublestranded cDNA molecule by I restriction sites of Sau3A to connector,Wherein X1 and X2,Including 30-37 nucleotide and difference,Including 20-25 blood pressure PCR prime sites and 55 °C -65 °C of Tm,( 6 )The product obtained in digestion step( 5 )With the enzyme BsmF I of label,The BsmF flush ends( 7 )From the ligation of the hybrid tag of I label of archaeal dna polymerase and different connectors,( 8 )It is obtained in binding markers step( 7 )To form double label DNA ligases,( 9 )Amplify obtained in a step and includes( 8 )With primer 2 0-25 blood pressures and with 55 ° -65 °C of Tm,( 10 )Double labels, which are isolated, has the product that blood pressure is obtained from amplification procedure between 20 and 28( 9 )Separation product is digested by the product and Sau3A I that digest the amplification,)250)251 connections,To form jugate obtained in the step,The jugate purifiying separation jugate has greatly ratio) 252 bp,)253 Hes)The different labels that the 254 jugate cloning and sequencing is analyzed.