Primer Extension Analysis to Map Transcription Start Sites of Vascular Genes
互联网
404
The principle of the technique presented in this chapter is illustrated in Fig. 1 . As with S1 mapping or riboprobe mapping, this technique can be used to determine precisely the start site of transcription of a mRNA sequence (
1
–
3
). Since this technique is relatively easier than other techniques, it is readily used for the primary determination of the transcription start site of a target gene. A radiolabeled probe derived entirely from within the gene is hybridized to mRNA complementary to the probe and extended using reverse transcriptase (RT). The cloned probe is normally derived from a region near the 5′ end (cap site) of the gene and the extension reaction terminates at the extreme 5′ end of the mRNA. Since only a small fragment of DNA probe is required as a primer, synthetic oligonucleotides are now almost exclusively used (although it is possible to use double-stranded DNA fragments or single-stranded primers generated by restriction enzyme digestion and gel electro-phoresis) (
4
).
Fig. 1.
The principle of primer extension analysis using a 5′ end-labeled probe. The diagram illustrates how the 5′ terminus of a poly(A)
+
mRNA might be determined using a 5′ end-labeled single-stranded DNA probe (or synthetic oligonucleotide). Usually the probe would be a fragment derived from a cloned copy of the gene by cleavage with appropriate restriction enzymes or an oligonucleotide synthesized from the known cDNA sequence. The schematic representation of the autoradiogram demonstrates the results expected from a typical experiment
Lane 1
, untreated probe;
lanes 2
and
3
, probe incubated under annealing conditions in the absence or presence of mRNA complementary to the probe, respectively, and then incubated with RT in the presence of unlabeled four deoxynucleoside trisphosfates (dNTPs). Only when complementary mRNA is present to form a hybrid with the primer, a primer extension product is synthesized as shown in lane 3.
