Processing

Please wait...

Settings

Settings

Goto Application

1. WO2002038809 - METHOD AND APPARATUS FOR DETECTING A MUTAITON IN A NUCLEIC ACID FRAGMENT IN A SAMPLE

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

CLAIMS

1. Method for detecting one or more mutation(s) in a nucleic acid fragment in a sample, comprising the

following steps, to be performed in suitable sequence, of:

(a) amplifying the nucleic acid fragment present in the sample;

(b) separating the nucleic acid fragments by means of gel electrophoresis in the presence of a gradient resulting in at least partial melting of the double-stranded nucleic acid fragments formed in step (a), for the purpose of fixing the partially melted nucleic acid fragments at a specific location in the gel; and

(c) detecting the separated nucleic acid fragments, characterized in that the amplification step (a) is performed in or on the gel.

2. Method as claimed in claim 1, characterized in that the method further comprises of:

(d) causing the double-stranded nucleic acid

fragments present in the sample to melt completely into single-stranded nucleic acid fragments prior to step (b), and reforming double-stranded fragments from these single-stranded nucleic acid fragments, wherein

heteroduplex double-stranded nucleic acid fragments are formed in addition to homoduplex double-stranded nucleic acid fragments.

3. Method as claimed in claim 1 or 2, characterized in that the gel electrophoresis is capillary gel

electrophoresis.

4. Method as claimed in claim 1, 2 or 3,

characterized in that the method further comprises of:

(e) changing the electrophoresis conditions after step (b) such that the at least partially melted nucleic acid fragments once again become double-stranded, whereby the separated nucleic acid fragments migrate further from their specific location in the gel at a practically equal speed.

5, Method as claimed in either of the claims 3 or 4, characterized in that the separated nucleic acid

fragments are detected when they leave the capillary.

6. Method as claimed in any of the foregoing claims 1-5, characterized in that the method further comprises of isolating the separated nucleic acid fragments from the gel.

7. Method as claimed in any of the foregoing claims 1-6, characterized in that the gradient in the gel resulting in at least partial melting of the double-stranded nucleic acid fragments is a temperature

gradient.

8. Method as claimed in any of the foregoing claims 1-6, characterized in that the gradient resulting in at least partial melting of the double-stranded nucleic acid fragments is a chemical gradient.

9. Method as claimed in claim 8, characterized in. that the chemical gradient is formed by urea and

formamide.

10. Method as claimed in claim 7, a or 9,

characterized in that the gradient consists of a

combination of a temperature gradient and a chemical gradient.

11. Method as claimed in any of the foregoing claims 2-10, characterized in that changing the electrophoresis conditions such that the at least partially melted nucleic acid fragments once again form double-stranded nucleic acid fragments comprises of reducing the

temperature in the gel.

12. Method as claimed in any of the foregoing claims 1-11, characterized in that the sample comprises genetic material from an individual wherein the genetic material is present in the form of nucleic acid, such as blood, sperm, saliva and/or tissue cells.

13. Device for detecting one or more mutation(s) in a nucleic acid fragment in a sample, comprising a number of capillaries in which a gel is arranged, wherein both the upper side and the underside of the gel in the capillaries are in contact with a liquid bath in which an electrode is arranged, a voltage source for applying a voltage over the gel, means for changing the

electrophoresis conditions during electrophoresis and means for detecting the separated DNA fragments.

14. Device as claimed in claim 13, characterized in that the upper side of the gel in the capillaries is in contact with one collective liquid bath for substantially all capillaries, wherein an electrode is arranged in the collective liquid bath, and the underside of each gel in the capillaries is in contact with separate liquid baths, wherein an electrode is arranged in each separate liquid bath.

15- Device as claimed in claim 13 or 14,

characterized in that the means for changing the

electrophoresis conditions in the capillaries are means for changing the temperature in the capillaries.

16. Device as claimed in claim 15, characterized in that the means for changing the temperature comprise a Peltier element.

17. Device as claimed in any of the foregoing claims 13-16, characterised in that means for measuring the temperature are arranged in at least one capillary.

18. Device as claimed in claim 17, characterized in that the means for measuring the temperature comprise a platinum resistance wire arranged in at least one capillary.

19. Device as claimed in any of the claims 13-18, characterized in that the capillaries are coated with primers and dNTPs for amplifying in the gel the nucleic acid fragments present in the sample,

20. Device as claimed in any of the claims 13-15, characterized in that the capillaries are further coated with polymerase for amplifying in the gel the nucleic acid fragments present in the sample.

21. Capillary for use in the method as claimed in any of the claims 1-12 and/or in the device as claimed in any of the claims 13-20, characterized in that the capillary is coated with primers and dNTPs.

22. Capillary as claimed in claim 21, characterized in that the capillary is further coated with polymerase.