Patent ID: 6225077
Filing Date: 2001-05-01
Classification: C12N,C12Q

Abstract:
A method for characterizing CDNA, which comprises:(a) cutting a sample comprising a population of one or more cDNAs or isolated fragments thereof, each having a strand complementary to the 3' poly-A terminus of an mRNA and bearing a tail, with a first sampling endonuclease at a first sampling site of known displacement from a reference site proximal to the tail to generate from each cDNA or isolated fragment thereof a first and second sub-fragment, each comprising a sticky end sequence of predetermined length and unknown sequence, the first sub-fragment bearing the tail;(b) sorting either the first or second sub-fragments into sub-populations according to their sticky end sequence and recording the sticky end sequence of each sub-population as the.first sticky end;(c) cutting the sub-fragments in each sub-population with a second sampling endonuclease, which is the same as or different from the first sampling endonuclease, at a second sampling site of known displacement from the first sampling site to generate from each sub-fragment a further sub-fragment comprising a second sticky end sequence of predetermined length and unknown sequence; and(d) determining each second sticky end sequence;wherein the aggregate length of the first and second sticky end sequences of each sub-fragment is from 6 to 10; and wherein the sequences and relative positions of the reference site and first and second sticky ends are utilized to characterize the cDNA or cPNAs.