包含的cDNA克隆数目较少.PPTVIP

Construction of a cDNA library reverse transcriptase makes a DNA copy of an RNA The life cycle of a retrovirus depends on reverse transcriptase retrovirus 1. virus enters cell and looses envelope 2. the capsid is uncoated, releasing genomic RNA and reverse transcriptase 3. reverse transcriptase makes a DNA copy 4. then copies the DNA strand to make it double-stranded DNA, removing the RNA with RNase H 5. the DNA is then integrated into the host cell genome where it is transcribed by host RNA polymerase II 6. it is translated into viral proteins, and assembled into new virus particles new viruses cDNA library construction AAAAA 5’ 3’ mRNA (all mRNAs in cell) anneal oligo(dT) primers of 12-18 bases in length AAAAA TTTTT 5’ 3’ 5’ add reverse transcriptase and dNTPs AAAAA TTTTT 5’ 3’ 3’ 5’ cDNA add RNaseH (specific for the RNA strand of an RNA-DNA hybrid) and carry out a partial digestion AA TTTTT 5’ short RNA fragments serve as primers for second strand synthesis using DNA polymerase I 3’ 3’ AAAAA TTTTT 5’ 3’ short RNA fragments serve as primers for second strand synthesis using DNA polymerase I AAA TTTTT 5’ 3’ DNA polymerase I removes the remaining RNA with its 5’ to 3’ exonuclease activity and continues synthesis DNA ligase seals the gaps AAA TTTTT 5’ 3’ AAAAA TTTTT 5’ 3’ double-stranded cDNA AAAAA TTTTT 5’ 3’ NNNNNNNNG NNNNNNNNCTTAA EcoRI linkers are ligated to both ends using DNA ligase AAAAANNNNNNNNG TTTTTNNNNNNNNCTTAA double-stranded cDNA copies of mRNA with EcoRI cohesive ends are now ready to ligate into a bacteriophage lambda vector cut with EcoRI 5’ 3’ AATTCNNNNNNNN GNNNNNNNN EcoRI sites combine cDNAs with lambda arms and treat with DNA ligase “left arm” “right arm” “left arm” “right arm” package into bacteriophage and infect E. coli 1 2 3 4 5 6 cDNA library in which each phage contains a different human cDNA 7 cDNAs cDNA Library The Central Dogma