DNA typing with short tandem repeat polymorphisms and identification of polymorphic short tandem repeats

• US 5,364,759 A
• Filed: 01/31/1991
• Issued: 11/15/1994
• Est. Priority Date: 01/31/1991
• Status: Expired due to Term

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Edwards, Albert O., Caskey, Charles T.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): JACOBSON, DIAN COOK
• Application Number:
  US07/647,655
• Time in Patent Office: 1,384 Days
• Field of Search: 435/6, 435/91, 436/94, 935/77, 935/78, 536/27, 536/24.3, 536/24.31, 536/24.33, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00574 radioactive
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00578 electrophoric
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2531/113 PCR
• 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 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a DNA profiling assay for detecting polymorphisms in a short tandem repeat. The method includes the steps of extracting DNA from a sample to be tested, amplifying the extracted DNA and identifying the amplified extension products for each different sequence. Each different sequence is differentially labeled. In the method, internal and external standards can also be used. The method is applicable to a wide variety of forensic and medical samples, including blood, semen, vaginal swaps, tissue, hair, saliva, urine and mixtures of body fluids. A short tandem repeat sequence which can be characterized by the formula (A_w G_x T_y C_z)_n, wherein A, G, T and C represent the nucleotides, w, x, y and z represent the number of nucleotide and range from 0 to 7 and the sum of w+x+y+z ranges from 3 to 7 and n represents the repeat number and ranges from 5 to 50. The labels can be from a variety of groups, including fluorescers, radioisotopes, chemiluminescers, stains, enzymes and antibodies. Also described is a kit. Further, a method of detecting the polymorphic short tandem repeats comprising the steps of either searching for the repeats in a data base or comparing oligonucleotides and searching for the repeats in a genetic library.

Claims

1. A DNA profiling assay for detecting polymorphisms in at least one short tandem repeat, comprising the steps of:

extracting DNA from a sample to be tested;

amplifying said at least one short tandem repeat in the extracted DNA, wherein the short tandem repeat sequence is characterized by the formula (A_w G_x T_y C_z)_n wherein A,G,T, and C represent the nucleotides;

w, x, y and z represent the number of each nucleotide and range from 0 to 7;

the sum of w+x+y+z ranges from 4 to 7; and

n represents the repeat number and ranges from about 5 to 50; and

detecting said polymorphisms by identifying said amplified extension products for each different sequence, wherein each different sequence is differentially labelled.

2. The method of claim 1, further comprising an external standard.

3. The method of claim 1, further comprising an internal standard.

4. The method of claim 1, wherein the sample to be tested is a forensic or medical sample selected from the group consisting of blood, semen, vaginal swabs, tissue, hair, saliva, urine, and mixtures of body fluids.

5. The assay of claim 1, wherein at least one short tandem repeat sequence is selected from the non-duplicative alphabetical represented nucleotide sequence group consisting of:

(AAAC)_n, (AAAG)_n, (AAAT)_n, (AACC)_n, (AACG)_n, (AAGC)_n, (AAGG)_n, (AAGT)_n, (AATC)_n, (AATG)_n, (AATT)_n, (ACAG)_n, (ACAT)_n, (AGAT)_n, (ACCC)_n, (ACCG)_n, (ACCT)_n, (ACGC)_n, (ACGG)_n, (ACGT)_n, (ACTC)_n, (ACTG)_n, (AGCC)_n, (AGCG)_n, (AGCT)_n, (AGGC)_n, (AGGG)_n, (ATCC)_n, (ATCG)_n, (ATGC)_n, (CCCG)_n, (CCGG)_n, and combinations thereof;

wherein n is the repeat number and varies from about 5 to 50.

6. The assay of claim 1 wherein n ranges between 5 and 40.

7. The assay of claim 1, wherein n varies from about 5 to 20.

8. The assay of claim 1, wherein the DNA is amplified by PCR or multiplex PCR.

9. The assay of claim 1, wherein amplification is by multiplex PCR with primers to at least two short tandem repeat sequences.

10. The assay of claim 1, wherein the label is selected from the group consisting of fluorescers, radioisotopes, chemiluminescers, stains, enzymes and antibodies.

11. The method of claim 10, wherein the label is fluorescent and is selected from the group consisting of Texas Red, NBD aminoheanoic acid Tetramethylrhodamine-5- (and -6) isothiocyanate, and Fluorescein-5-isothiocyanate.

12. The DNA profiling assay of claim 1, further comprising the step of distinguishing simultaneously differential labels during the detecting step, wherein said labels are distinguished by an automated DNA label analyzer.

22. The assay of claim 8, wherein the primer pairs are selected from the group consisting of SEQ ID. NO. 1 and 2, SEQ ID. NO. 3 and 4, SEQ ID. NO. 5 and 6, SEQ ID. NO. 7 and 8, SEQ ID. NO. 9 and 10, SEQ ID. NO. 11 and 12, SEQ ID. NO. 13 and 14, SEQ ID. NO. 15 and 16, SEQ ID. NO. 17 and 18, SEQ ID. NO. 19 and 20, SEQ ID. NO. 21 and 22, SEQ ID. NO. 5 and 24, SEQ ID. NO. 19 and 25 and Seq. ID NO. 13 and 23.

25. The assay of claim 1, wherein the short tandem repeat sequence further includes sequences selected from the non-duplicative alphabetical represented nucleotide sequence group consisting of (AAC)_n, (AAG)_n, (AAT)_n, (ACC)_n, (ACG)_n, (ACT)_n (AGC)_n, (AGG)_n, (ATC)_n, and (CCG)_n.

13. A kit for a DNA profiling assay to detect polymorphisms in at least one short tandom repeat sequence comprising a container which has oligonucleotide primer pairs for amplifying said at least one short tandem repeat sequence.

14. The kit of claim 13 further comprising a labelled standard.

15. The kit of claim 13 further comprising a container having reagents for multiplex polymerase chain reaction.

23. The assay of claim 13, wherein the primer pairs are selected from the group consisting of SEQ ID. o NO. 1 and 2, SEQ ID. NO. 3 and 4, SEQ ID. NO. 5 and 6, SEQ ID. NO. 7 and 8, SEQ ID. NO. 9 and 10, SEQ ID. NO. 11 and 12, SEQ ID. NO. 13 and 14, SEQ ID. NO. 15 and 16, SEQ ID. NO. 17 and 18, SEQ ID. NO. 19 and 20, SEQ ID. NO. 21 and 22, SEQ ID. NO. 5 and 23, SEQ ID. NO. 19 and 24.

16. A method of detecting a polymorphic short tandem repeat comprising the steps of:

determining possible, non-duplicative nucleotide sequences of the formula (A_w G_x T_y C_z), wherein A,G,T and C represents each nucleotide and w,x,y and z represent the number of each nucleotide and ranges between 0 and 7 with the sum of w+x+y+z ranging between 3 and 7;

searching for (A_w G_x T_y C_z)_n in databases containing known genetic sequences, wherein n represents the number of tandem repeats of the genetic sequence and is at least 5;

identifying the (A_w G_x T_y C_z)_n sequence and its flanking sequence;

extracting each identified sequence found in the searching step and its flanking sequence; and

identifying the extracted sequences which have unique flanking sequences.

17. The method of claim 16, further comprising the steps of:

synthesizing oligonucleotide primer pairs to the unique flanking sequences;

performing a polymerase chain reaction with the primer pairs on DNA samples from a test population; and

detecting polymorphic short tandem repeats in the extension products of the polymerase chain reaction.

18. A method of detecting a polymorphic short tandem repeat comprising the steps of:

synthesizing labelled oligonucleotide probes complementary to said short tandem repeat;

hybridizing the labelled probes to total human λ

phage libraries; and

sequencing the hybridized plaques.

19. The method of claim 18, wherein the sequencing step, includes the step of subcloning the hybridized plaque.

20. The method of claim 18, wherein the sequencing step, includes the step of direct polymerase chain reaction of the hybridized plaque.

21. The short tandem AGAT repeat as defined in SEQ ID. NO. 26.

24. The sequences of SEQ ID. NO. 1, SEQ ID. NO. 2, SEQ ID. NO. 3, and SEQ ID. NO. 4 for determining flanking sequences of short tandem repeat.