Simultaneous amplification of multiple targets

• US 5,624,825 A
• Filed: 05/26/1995
• Issued: 04/29/1997
• Est. Priority Date: 06/04/1993
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Walker, George T., Little, Michael C., Nadeau, James G.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US08/451,313
• Time in Patent Office: 704 Days
• Field of Search: 435/6, 435/91.2, 435/91.5, 935/17, 935/77, 935/78, 536/24.32, 536/24.33, 536/24.3
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: G11B 19/04 Arrangements for preventing, inhibiting, or warning against double recording on the same blank or against other recording or reproducing malfunctions
• CPC Class Codes: G11B 2005/0008 Magnetic conditionning of heads, e.g. biasing
• CPC Class Codes: G11B 5/09 Digital recording
• CPC Class Codes: G11B 5/1878 disposed immediately adjacent to the transducing gap, e.g. "Metal-In-Gap" structure
• CPC Class Codes: G11B 5/3113 for improving the magnetic domain structure or avoiding the formation or displacement of undesirable magnetic domains
• CPC Class Codes: G11B 5/455 Arrangements for functional testing of heads; Measuring arrangements for heads
• CPC Class Codes: G11B 5/465 Arrangements for demagnetisation of heads

Abstract

Methods for multiplex amplification of target nucleic acid sequences using a single pair of primers. Defined sequences are appended to the ends of multiple target sequences as part of the amplification reaction so that no steps in addition to amplification are required. The target sequences with the appended defined sequences need not be isolated prior to amplification. In one embodiment for coamplification of two target sequences, a sequence corresponding to a terminal segment of the first target sequence is appended to one end of the second target sequence and a sequence corresponding to a terminal segment of the second target sequence is appended to one end of the first target sequence. Amplification of the two targets then requires only a single pair of primers. Alternatively, a single defined sequence may be appended to the 5'"'"' and 3'"'"' ends of any number of selected targets. All such modified target sequences may then be amplified using a single pair of primers which hybridize to the defined end-sequences.

Claims

1. A method for simultaneously amplifying a first and a second target nucleic acid sequence by Strand Displacement Amplification (SDA) comprising:

a) hybridizing a first adapter primer to the 3'"'"' end of the first target sequence, the 3'"'"' end of the adapter primer comprising a target binding sequence which hybridizes to the target sequence and the 5'"'"' end of the adapter primer comprising a first adapter sequence which is substantially identical to a 3'"'"' target binding sequence of a first amplification primer which hybridizes to a first strand of the second target sequence;

b) extending the first adapter primer with polymerase to produce a first extension product which is complementary to the first target sequence and displacing the first extension product;

c) hybridizing a second adapter primer to the first extension product at the 3'"'"' end of the complement of the first target sequence, the 3'"'"' end of the second adapter primer comprising a target binding sequence which hybridizes to the first extension product and the 5'"'"' end of the second adapter primer comprising a second adapter sequence which is substantially identical to a 3'"'"' target binding sequence of a second amplification primer which hybridizes to a second strand of the second target sequence;

d) extending the second adapter primer with polymerase to produce a second extension product, displacing the second extension product, and;

e) simultaneously amplifying the second extension product and the second target sequence in an SDA reaction using the first and second amplification primers, the first and second amplification primers each further comprising a recognition site for a restriction enzyme which nicks one strand of a hemimodified double stranded restriction enzyme recognition site, thereby amplifying the first and second target nucleic acid sequence.

2. The method according to claim 1 wherein the first and second extension products are displaced by extension of bumper primers.

3. The method according to claim 2 wherein the amplification primers are present in approximately 10-fold excess over the first and second adapter primers.

4. The method according to claim 3 wherein the first and second target nucleic acid sequences are the IS6110 insertion element of Mycobacterium tuberculosis and the 16S ribosomal gene of Mycobacterium tuberculosis.

5. The method according to claim 4 wherein the first and second target nucleic acid sequences are the IS6110 insertion element of Mycobacterium tuberculosis and a target sequence from Mycobacterium avium.

6. The method according to claim 1 wherein the second extension products are amplified in an SDA reaction which employs a restriction enzyme selected from the group consisting of HincII, HindII, AvaI, NciI, and Fnu4HI for nicking the hemimodified restriction enzyme recognition site.

7. The method according to claim 1 further comprising the steps of:

a) hybridizing the second adapter primer to the 3'"'"' end of a second strand of the first target sequence, extending the second adapter primer to form a third extension product complementary to the second strand of the first target sequence, and displacing the third extension product;

b) hybridizing the first adapter primer to the third extension product at the 3'"'"' end of the complement of the first target sequence, extending the first adapter primer to form a fourth extension product, and displacing the fourth extension product, and;

c) simultaneously amplifying the fourth extension product and the second target sequence in the SDA reaction using the first and second amplification primers.

8. A method for simultaneously amplifying a first and a second target nucleic acid sequence in a primer extension amplification reaction comprising:

a) hybridizing a first adapter primer to the 3'"'"' end of the first target sequence, the 3'"'"' end of the adapter primer comprising a target binding sequence which hybridizes to the target sequence and the 5'"'"' end of the adapter primer comprising a first adapter sequence which is substantially identical to a 3'"'"' target binding sequence of a first amplification primer which hyridizes to a first strand of the second target sequence;

b) extending the first adapter primer with polymerase to produce a first extension product which is complementary to the first target sequence and displacing the first extension product;

c) hybridizing a second adapter primer to the first extension product at the 3'"'"' end of the complement of the first target sequence, the 3'"'"' end of the second adapter primer comprising a target binding sequence which hybridizes to the first extension product and the 5'"'"' end of the second adapter primer comprising a second adapter sequence which is substantially identical to a 3'"'"' target binding sequence of a second amplification primer which hybridizes to a second strand of the second target sequence;

d) extending the second adapter primer with polymerase to produce a second extension product, displacing the second extension product, and;

e) simultaneously amplifying the second extension product and the second target sequence in a primer extension amplification reaction using the first and second amplification primers thereby amplifying the first and second target nucleic acid sequence.

9. The method according to claim 8 further comprising appending adapter sequences to a third target nucleic acid sequence according to steps (a) through (d) and simultaneously amplifying the first, second and third target sequences using the first and second amplification primers.