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Oligonucleotide sizing using immobilized cleavable primers

  • US 5,700,642 A
  • Filed: 05/22/1995
  • Issued: 12/23/1997
  • Est. Priority Date: 05/22/1995
  • Status: Expired due to Term
First Claim
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1. A method for determining the size of a primer extension product, comprising(a) hybridizing a primer with a target nucleic acid, where said primer (i) is complementary to said target nucleic acid;

  • (ii) has a first region containing the 5'"'"' end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3'"'"' end of the primer, where the 3'"'"' end is capable of serving as a priming site for enzymatic extension and where said second region contains a selected cleavable site,(b) extending the primer enzymatically to generate a polynucleotide mixture containing an extension product composed of the primer and an extension segment;

    (c) cleaving said extension product at the cleavable site to release said extension segment, where prior to said cleaving the primer is immobilized at said immobilization attachment site; and

    (d) sizing the extension segment by mass spectrometry, whereby said cleaving is effective to increase the read length of the extension segment relative to the read length of the product of (b).

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