Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products

DETAILED DESCRIPTION OF THE INVENTION Applicants' invention provides a simplified method for isolating primer extension products from template-directed DNA polymerase reactions.
For the purposes of this application, the following terms and phrases are important to an understanding of the invention: "Primer" means a single stranded oligonucleotide capable of hybridizing at one or more specific locations or "priming sites" in the template nucleic acid.
"Primer extension product" means a primer to which one or more naturally occurring or modified nucleotides have been added by template directed enzymatic addition to the 3' end of the primer.
The process requires hybridization of the primer to the template.
"Biotinylated primer" means a primer covalently linked to a biotin or an analog of biotin.
The linking group used should permit enzymatic primer extension and hybridization between the primer and the template.
The binding of biotin and analogs of biotin to avidin is reviewed by Green (Advances in Protein Chemistry, 29, 85-133, 1975).
"Template" means a single or double stranded nucleic acid to be analyzed by means of primer extension reactions.
"Template-directed polymerase reaction" or "primer extension reaction" includes, but is not limited to, a standard Sanger sequencing reaction (Sanger et al.
, Proc.
Natl.
Acad.
Sci.
U.
S.
A.
, 74, 5463-5467, 1977), a fluorescent terminator sequencing reaction (Prober et al.
, Science, 238, 336-341, 1987), PCR (Saiki et al.
, Science, 230, 1350-1355, 1985), or some other template-directed primer extension reaction such as ligase-mediated gene detection (Landegren et al.
, Science, 241, 1077-1081, 1988).
FIG.
1 illustrates one embodiment of Applicants' invention and FIG.
2 illustrates a second embodiment.
The only difference between these two processes occurs in step 2, wherein a different method is used to separate template from biotinylated primer extension products.
In fact, as shown in the figures and described more fully below, these operational differences result in differences in the structure of the captured nucleic acids