POLYNUCLEOTIDE SEPARATION METHOD

Abstract

Various method of isolating a modified polynucleotide having the formula A-P-B from a crude mixture of modified polynucleotides also including those having the formulae A-P-A and B-P-B, wherein P is a polynucleotide region and A and B are different modifying moieties or nucleotide sequences are provided. The methods include the steps of (a) reacting the crude mixture concurrently or consecutively with beads of one type capable of binding to moiety A but not B, and beads of a second type capable of binding to moiety B but not A; (b) fractionating the intermediate product based on the properties of each type of bead and of pairs of different types of bead conjoined by A-P-B polynucleotides, such that only or predominantly conjoined pairs of the two different types of bead are retained and (c) optionally, releasing one or both beads from such conjoined pairs such that the polynucleotide may be recovered.

Claims

1. A method of isolating a modified polynucleotide having the formula A-P-B from a crude mixture of modified polynucleotides also including those having the formulae A-P-A and B-P-B, wherein P is a polynucleotide region and A and B are different modifying moieties or nucleotide sequences, comprising the steps of: (a) reacting the crude mixture with first beads modified with complementary moieties A capable of binding to A and second beads having a density lower than that of the first beads and modified with complementary moieties B capable of binding to B to produce an intermediate product comprised of first-second bead-pairs connected by a modified polynucleotide together with one or more of unused or unpaired first and second beads and corresponding first-first, second-second bead-pairs; (b) separating the first-second bead-pairs and optionally any unused or unpaired second beads or second-second bead-pairs from the intermediate product in an aqueous medium having a density greater than that of the first-second beads-pairs but less than that of the first beads and (c) thereafter separating the first-second bead-pairs from the product of step (b) in an aqueous medium having a density less than that of the first-second bead-pairs but greater than that of the second beads.

2. The method of claim 1 wherein the first beads are magnetic.

3. A method of isolating a modified polynucleotide having the formula A-P-B from a crude mixture of modified polynucleotides also including those having the formulae A-P-A and B-P-B, wherein P is a polynucleotide region and A and B are different modifying moieties or nucleotide sequences comprising the steps of: (a) reacting the crude mixture with magnetic first beads modified with complementary moieties A capable of binding to A and non-magnetic second beads having a density lower than that of the first beads and modified with complementary moieties B capable of binding to B to produce an intermediate product comprised of first-second bead-pairs connected by a modified polynucleotide together with one or more of unused or unpaired first and second beads and corresponding first-first, second-second bead-pairs; (b) separating the first-second bead-pairs and optionally any unused and/or unpaired second beads and/or second-second bead-pairs from the intermediate product in a liquid medium having a density greater than that of the first-second beads-pairs and (c) thereafter in an aqueous medium separating the first-second bead-pairs from the product of step (b) by applying a magnetic field sufficiently strong to overcome the buoyancy of the first-second bead pairs in the liquid medium.

4. A method of isolating a modified polynucleotide having the formula A-P-B from a crude mixture of modified polynucleotides also including those having the formulae A-P-A and B-P-B, wherein P is a polynucleotide region and A and B are different modifying moieties or nucleotide sequences comprising the steps of: (a) reacting the crude mixture with magnetic first beads modified with complementary moieties A capable of binding to A and non-magnetic second beads having a density lower than that of the first beads and modified with complementary moieties B capable of binding to B to produce an intermediate product comprised of first-second bead-pairs connected by a modified polynucleotide together with one or more of unused or unpaired first and second beads and corresponding first-first, second-second bead-pairs; (b) separating the first-second bead pairs and optionally any unused and/or unpaired first beads and/or first-first bead pairs from the intermediate product in a liquid medium by the application of a magnetic field and (c) thereafter separating the first-second bead-pairs from the product of step (b) in a liquid medium having a density greater than that of the first-second bead-pairs but greater than the second beads.

5. The method of claim 1, wherein step (a) comprises two sub-steps in which the crude mixture is either first treated with the first beads and thereafter the second beads or first treated with the second beads and thereafter the first beads.

6. The method of claim 1, wherein the densities of the liquid media are controlled or modified by use of a Group IA or IIA soluble salt or a sugar.

7. The method of claim 1, wherein the density of the liquid medium in step (b) is 100-120% of the density of the first-second bead pairs.

8. The method of claim 1, wherein the density of the liquid medium in step (c) is 75-90% of the density of the first-second bead pairs.

9. The method of claim 1, wherein the first beads have a density of greater than 1.5 g/cm.sup.3 and the second beads have a density of less than 1.5 g/cm.sup.3.

10. The method of claim 1, wherein at least one of the pairs A and A or B and B are selected from avidin/biotin, streptavidin/biotin, a protein/antibody pair or two complementary oligonucleotide regions.

11. The method of claim 1, further comprising step (d) detaching the beads from the first-second bead pair.

12. The method of claim 1, wherein the modified polynucleotide or at least the P moiety thereof is a ribonucleotide, a deoxyribonucleotide or a synthetic analogue thereof.

13. The method of claim 1 wherein the polynucleotide is isolated for subsequent sequencing.

Description

EXAMPLE

[0035] In this example the non-magnetic beads are polystyrene beads (density .sup.1.05) having a radius of 2 m, and functionalized with an anti-digoxigenin (Anti-DIG) antibody (Spherotech, product DIG-40-2). The magnetic beads (density .sup.1.8) have a radius of 0.5 m and are functionalized with streptavidin (Dynal MyOne T1 streptavidin beads).

[0036] Polynucleotide fragments of 10 kb were prepared and their 3 and 5 ends labelled with a mixture of digoxigenin (DIG) and biotin (BIO), such that each end of a given fragment is equally likely to carry either a DIG or a BIO moiety. Hence, the population of modified fragments contain approximately 25% DIG--, 25% BIO--, and 50% DIG--BIO (=BIO-DIG) fragments. This crude mixture of modified polynucleotides was then diluted to a concentration of 11 pg/l, equivalent to approximately 10.sup.6 molecules/l.

[0037] 5 l of Dynal MyOne T1 beads (containing approximately 510.sup.7 beads) were then washed into 200 l of Dynal binding buffer according to the manufacturer's instructions, and 5 l of the crude mixture (approximately 510.sup.6 molecules) then added. This mixture was then placed on a tube rotator at 22 C. for 30 minutes to allow beads to bind to any modified polynucleotides carrying BIO. The beads were then washed, following the manufacturer's instructions, to remove any unbound polynucleotide (specifically, molecules labelled at both ends with DIG which will not have bound to Dynal beads), and the beads then recovered into 10 microlitres of Link Buffer (lx Phosphate Buffered Saline, 0.1% Tween 20 detergent).

[0038] 0.5 ml of the non-magnetic Anti-DIG beads (approximately 510.sup.7 beads) were washed by centrifugation into 10 l of Link Buffer and added to a suspension of the washed magnetic beads (total volume=20 l). This mixture was again placed on a tube rotator at 22 C. for 60 minutes to allow the non-magnetic beads to bind to any DNA polynucleotides carrying DIG.

[0039] At the end of this period a 130 ul of wash buffer was added to the beads suspension and then it was further diluted with 280 ul of a 1.9M sucrose solution (density 1.24), bringing the overall density of the solution to 1.17. The resulting mixture was then centrifuged at 800 G for 30 minutes. This caused any used free Dynal beads (density 1.8) and any modified polynucleotide linking two Dynal beads to sink and those linked by one Dynal bead any non-magnetic bead to be driven to the top of the liquid. The sunk pellets were then removed by aspiration using a pipette, and discarded, leaving the upper part of the liquid undisturbed. Finally, the remaining upper layer of the liquid was removed and transferred to a new tube. The contents of this new tube comprised free non-magnetic beads and Dynal/Anti-DIG bead pairs linked by a modified polynucleotide having one BIO and one DIG end.

[0040] A magnetic field was then applied to a wall of the new tube for 1 minute causing the Dynal/Anti-DIG bead pairs to be drawn to the wall and thus separated. Thereafter, the remaining liquid was aspirated out of the tube, and the magnetic field removed. 500 l of wash buffer (10 mM Tris HCl (pH7.5), 1 mM EDTA) was then added, and the remaining bead-pairs gently re-suspended. The magnetic field was then re-applied for a further one minute to once again draw the Dynal/Anti-DIG bead pairs to the wall of the tube before the liquid was again aspirated away and the magnetic field removed. Finally, the bead-pairs were recovered and re-suspended in 100 l of wash buffer prior to the beads being removed from the polynucleotide.

[0041] Further methods of effecting the separation are set out schematically in the Figure.