Method for capturing long polynucleotides for sequencing specific gnomic regions.

摘要

The invention relates to methods for capturing a polynucleotide comprising a DNA target segment from a population of polynucleotide molecules. The method may use a mixture comprising the population of the polynucleotide molecules, one or more polynucleotide primers, nucleotides, biotinylated nucleotides, a polymerase enzyme and a buffered liquid medium. The method may denature the polynucleotide molecules in the mixture so as to create single stranded portions of the polynucleotide molecules which can be accessible to the polynucleotide primers; hybridizing the polynucleotide primers to the polynucleotide comprising the DNA target segment. The method may create an extended polynucleotide primer by using the nucleotides, the biotinylated nucleotides and the polymerase enzyme as a means for labeling with biotin and increasing the extent of hybridization of the extended polynucleotide primer to the polynucleotide comprising the DNA target segment. The method may add streptavidin-labeled magnetic beads to the mixture. The method may use a magnetic field to move the streptavidin-labeled magnetic beads relative to the buffered liquid medium as a means for increasing binding of the biotinylated nucleotides of the extended polynucleotide primer to the streptavidin-labeled magnetic beads. The method may use either a rotation of the magnetic field relative to the mixture or a rotation of the mixture relative to the magnetic field for causing a streptavidin-labeled magnetic bead rotation in the buffered liquid medium as a means for performing the steps of increasing the binding of the biotinylated nucleotides of the extended polynucleotide primer to the streptavidin-labeled magnetic beads and increasing the winding of the biotinylated nucleotides of the extended polynucleotide primer onto the streptavidin-labeled magnetic beads, so as to increase the probability of capturing a long polynucleotide comprising a DNA target segment and so as to increase the probability of preventing damage to the long polynucleotide comprising a DNA target segment. Also the method may use a magnetic field to collect the streptavidin-labeled magnetic beads so as to capture the polynucleotide comprising the DNA target segment which is bound to the streptavidin-labeled magnetic beads.