A new approach to template purification for sequencing applications using paramagnetic particles.
A new method using traditional hybridization methodology, coupled with the new magnetic particle technology, has been developed for DNA purification, specifically for sequencing applications. The method is similar to the reverse hybridization blot system; however, a specific oligonucleotide probe was attached to the paramagnetic particle instead of a sheet membrane. The target DNA containing the complementary sequence of the probe hybridizes to the probe that is attached to the bead and is then magnetically removed from solution, washed and collected. This system eliminates the need of organic extractions and precipitation/concentration steps. The entire hybridization-purification system can be done in a 1.5-ml microcentrifuge tube making the method ideal for automation. M13 phage clones were purified with this method, both by manual means and by using the CATALYST 800 Molecular Biology LabStation fitted with a prototype magnetic station, and then sequenced. DNA sequencing results obtained with this system were reproducible and gave excellent length of read with low background.
Fry
G
G
Applied Biosystems, Inc., Foster City, CA 94404.
Lachenmeier
E
E
Mayrand
E
E
Giusti
B
B
Fisher
J
J
Johnston-Dow
L
L
Cathcart
R
R
Finne
E
E
Kilaas
L
L
D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D015342:DNA Probes; D004279:DNA, Viral / Q000302:isolation & purification; D005821:Genetic Techniques; D007763:Lac Operon; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D013698:Templates, Genetic
D015342:DNA Probes; D004279:DNA, Viral; D004247:DNA