Methods and systems for molecular fingerprinting

• US 20070141599A1
• Filed: 10/17/2006
• Published: 06/21/2007
• Est. Priority Date: 09/23/1997
• Status: Abandoned Application

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen, Chou, Hou-Pu
• Application Number:
  US11/582,838
• Publication Number:
  US 20070141599A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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. A method of claim 1 wherein each fragment is selected at random.

3. A method of claim 2 wherein each fragment is about 20 to about 50 base pairs in length.

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. A method of claim 4, wherein the polynucleotide sample is digested before the combining step.

6. A method of claim 5, wherein digestion is performed with a digestion enzyme.

7. A method of claim 6, wherein the digestion enzyme is a six-base cutter.

8. A method of claim 5, wherein the polynucleotide sample is digested into fragments of tens of thousands of base pairs.

9. A method of claim 4 wherein the nucleotide triphosphates are fluorescently labeled.

10. A method of claim 4, wherein the sample fragments are compared to the fingerprint by determining the sizes of the sample fragments in relation to the fingerprint fragments.

11. A method according to claim 10 wherein determining the sizes of fragments comprises gel electrophoresis.

12. A method according to claim 10 wherein determining the sizes of fragments comprises discrimination in a microfluidic device.

13. A method of claim 4, wherein the polynucleotide sample is digested after the combining step.

14. A method according to claim 4, wherein the sample polynucleotide is a forensic sample.

15. A method according to claim 4, wherein the target polynucleotide is associated with a disease.

16-55. -55. (canceled)