Methods and Systems for Molecular Fingerprinting

• US 20100196892A1
• Filed: 10/09/2009
• Published: 08/05/2010
• Est. Priority Date: 09/23/1997
• Status: Abandoned Application

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Chou, Hou-Pu, Quake, Stephen R.
• Application Number:
  US12/577,078
• Publication Number:
  US 20100196892A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0484 Cantilevers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0633 with moving parts
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties

Abstract

This invention relates in general to a method for molecular fingerprinting. The method can be used for forensic identification (e.g. DNA fingerprinting, especially by VNTR), bacterial typing, and human/animal pathogen diagnosis. More particularly, molecules such as polynucleotides (e.g. DNA) can be assessed or sorted by size in a microfabricated device that analyzes the polynucleotides according to restriction fragment length polymorphism. In a microfabricated device according to the invention, DNA fragments or other molecules can be rapidly and accurately typed using relatively small samples, by measuring for example the signal of an optically-detectable (e.g., fluorescent) reporter associated with the polynucleotide fragments.

Claims

1. A molecular fingerprinting method comprising the steps of:

(a) identifying a target polynucleotide, (b) selecting at least one fragment of the target polynucleotide, wherein the fragment is a fixed distance from a restriction site, to generate a set of one or more polynucleotide fragments, and (c) designating some or all of the set of fragments as molecular fingerprint corresponding to the target polynucleotide.

2-3. -3. (canceled)

4. A method for identifying a polynucleotide sample comprising the steps of:

(a) identifying a target polynucleotide;

(b) selecting at least one fragment of the target polynucleotide, wherein the fragment is a fixed distance from a restriction site, to generate a set of one or more polynucleotide fragments;

(c) designating some or all of the set of fragments as a fingerprint corresponding to the target polynucleotide;

(d) synthesizing one or more oligonucleotide probes to complement the set of polynucleotide fragments;

(e) combining the probes, a polynucleotide sample, nucleotide triphosphates, and polymerase to synthesize at least one polynucleotide strand;

(f) cutting the strands with restriction enzymes to yield a set of sample fragments of fixed length; and

(g) comparing the set of sample fragments to the fingerprint.

5-15. -15. (canceled)

16. A method for detecting a particular nucleic acid in a sample, which particular nucleic acid has at least one restriction site, and which method comprises:

(a) contacting the sample with a primer that hybridizes to the particular nucleic acid a predetermined distance from the restriction site, a polymerase and a plurality of nucleotides, so that a complementary nucleic acid is synthesized from the primer at least to the restriction site;

(b) contacting the complementary nucleic acid with a restriction enzyme under conditions capable of cutting the complementary nucleic acid at the restriction site; and

(c) detecting a nucleic acid fragment having a particular length equal to the fixed distance, wherein the presence of the nucleic acid fragment in the sample indicates that the particular nucleic acid is present in the sample.

17-35. -35. (canceled)

36. A method for detecting a particular nucleic acid in a sample, which particular nucleic acid has at least one restriction site, and which method comprises:

(a) contacting the sample with a restriction enzyme under conditions capable of cutting the particular nucleic acid at the restriction site;

(b) contacting the sample with a primer that hybridizes to the nucleic acid a predetermined distance from the cut at the restriction site, a polymerase and a plurality of nucleotides, so that a complementary nucleic acid fragment is synthesized; and

(c) detecting the complementary to nucleic acid fragment, wherein the presence of the complementary nucleic acid fragment in the sample indicates that the particular nucleic acid is present in the sample.

37-55. -55. (canceled)