幻灯片1-biolab.szu.edu.cn.ppt

1.Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose 2.More rapid procedure is to add oligo(dT) linked to magnetic beads directly to a cell lysate and ‘pulling out’ the mRNA using a strong magnet 3.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradients Three methods to isolate mRNA. cDNA libraries Make sure that the mRNA is not degraded. Methods: Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translated Analysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels Check the mRNA integrity cDNA libraries Cloning the particular mRNAs Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library. Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gels Enrichment: carried out by hybridization Example: clone the hormone induced mRNAs (substrated cDNA library) cDNA libraries Synthesis of cDNA : First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1) Second strand synthesis: best way of making full-length cDNA is to employs a ribonuclease ( RNase H ) which recognises the RNA component of a DNA:RNA hybrid and cleaves the RNA at a number of non-specific sites .(Fig2) cDNA libraries Fig.1 First strand synthesis Fig.2 Second strand synthesis Treatment of cDNA ends Blunt end ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning. The process : Move protruding 3’-ends(strand-special nuclease) Fill in missing 3’ nucleotide (klenow fragment of DNA polyI and 4 dNTPs) Ligate the blunt-end and linkers(T4 DNA ligase) Restriction enzyme digestion (E.coRI ) Tailing wit