Oligonucleotide sizing using immobilized cleavable primers

A method for determining the size of a primer extension product, comprising

• (a) combining first and second primers with a target nucleic acid under conditions that promote the hybridization of the primers to the nucleic acid, thus generating primer/nucleic acid complexes, where said first 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 cleavable site, and where said second primer is homologous to said target nucleic acid,
• (b) converting the primer/nucleic acid complexes to double-stranded fragments in the presence of a suitable polymerase and all four dNTPs,
• (c) amplifying the primer-containing fragments by successively repeating the steps of (i) denaturing the double-stranded fragments to produce single-strand fragments, (ii) hybridizing the single strands with the primers to form strand/primer complexes, (iii) generating double-stranded fragments from the strand/primer complexes in the presence of DNA polymerase and all four dNTPs, and (iv) repeating steps (i) to (iii) until a desired degree of amplification has been achieved,
• (d) denaturing the amplified fragments to generate a mixture including a product composed of the first primer and an extension segment;
• (e) immobilizing amplified fragments containing the first primer, utilizing said immobilization attachment site, and removing non-immobilized amplified fragments,
• (f) cleaving said immobilized fragments at the cleavable site to release the extension segment;
• and
• (g) 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 (d).

A method for determining the DNA sequence of a target DNA sequence, comprising

• (a) hybridizing a primer with a target DNA, where said primer (i) is complementary to said target DNA;
• (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 cleavable site,
• (b) extending the primer with an enzyme in the presence of a first of four different dideoxy nucleotides to generate a mixture of primer extension products each product containing a primer and an extension segment;
• (c) cleaving at the cleavable site to release the extension segments, where prior to said cleaving the primers are immobilized at said immobilization attachment sites;
• (d) sizing the extension segments 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),
• (e) repeating steps (a) through (d) with a second, third, and fourth of the four different dideoxy nucleotides, and
• (f) determining the DNA sequence of said target DNA by comparison of the sizes of the extension segments obtained from each of the four extension reactions.

A method for determining the DNA sequence of a target DNA sequence, comprising

• (a) hybridizing a primer with a target DNA, where said primer (i) is complementary to said target DNA;
• (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 cleavable site,
• (b) extending the primer with an enzyme in the presence of a first of four different deoxynucleoside .alpha.-thiotriphosphate analogs (dNTP.alpha.S) to generate a mixture of primer extension products containing phosphorothioate linkages,
• (c) treating the primer extension products with a reagent that cleaves specifically at the phosphorothioate linkages,
• where said treating is carried out under conditions producing limited cleavage, resulting in the production of a group of primer extension degradation products,
• (d) washing the primer extension degradation products, where prior to said washing, the primer extension degradation products are immobilized at said immobilization attachment sites, each immobilized primer extension degradation product containing a primer and an extension segment, where said washing is effective to remove non-immobilized species,
• (e) cleaving at the cleavable site to release the extension segments,
• (f) sizing the extension segments by mass spectrometry, whereby said cleaving is effective to increase the read length of any given extension segment relative to the read length of its corresponding primer extension degradation product,
• (g) repeating steps (a) through (f) with a second, third, and fourth of the four different dNTP.alpha.Ss, and
• (h) determining the DNA sequence of said target DNA by comparison of the sizes of the extension segments obtained from each of the four extension reactions.