Apparatus and method for shear breakage of polynucleotides
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsPeter Josef Oefner, Scott P. Hunicke-Smith
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Abstract
Apparatus and method for producing a defined size distribution of polynucleotide fragments having a selected mean size range between about 100 and 20,000 base pairs. The method includes passing a suspension of polynucleotide fragments through an apparatus containing a capillary with an orifice near the outlet end of the capillary.
Claims26
An apparatus suitable for producing a defined size distribution of polynucleotide fragments in a polynucleotide suspension, said apparatus comprising:
- (i) a shearing assembly, said assembly comprising:
- (a) a capillary,
- (b) an orifice plate disposed within said capillary, said plate having an orifice with a diameter greater than about 0.001" and less than about 0.003", and
- (c) a port for injection of a polynucleotide suspension;
- (ii) a pumping means effective to pass said polynucleotide suspension through said shearing assembly at a flow rate of at least about 1 ml/minute in a manner that does not generate air bubbles within the fluid suspension;
- and
- (iii) a repeat pass means effective to pass said polynucleotide suspension through said shearing assembly a plurality of times,
- wherein said defined size distribution of polynucleotide fragments has a size range with a selected mean between about 100 and 20,000 bp, and where the largest polynucleotide fragment in the size distribution is about twice as large as the smallest polynucleotide fragment in the size distribution.
A method of producing a defined size distribution of sheared polynucleotide fragments having a size range with a selected mean between about 100 and 20,000 bp, and where the largest polynucleotide fragment in said size range is about twice as large as the smallest polynucleotide fragment in said size range, comprising:
- (i) passing a polynucleotide suspension through a shearing assembly at a flow rate of between 1 ml/min and 23 ml/min, said shearing assembly comprising:
- (a) a capillary,
- (b) an orifice plate disposed within said capillary, said plate having an orifice with a diameter of less than 0.005", and
- (c) a port for injection of said polynucleotide suspension,
- wherein said passing results in shearing polynucleotides in the suspension into sheared polynucleotide fragments;
- and
- (ii) repeating step (i) until said sheared polynucleotide fragments have selected mean between about 100 and 20,000 bp, and the largest polynucleotide fragment in said size range is about twice as large as the smallest polynucleotide fragment in said size range.
Dependencies are inferred from the claim text.