WO1994019480A1

WO1994019480A1 - Dna sequencing with non-radioactive label

Info

Publication number: WO1994019480A1
Application number: PCT/US1994/002028
Authority: WO
Inventors: Parke K. Flick; Barry Nash
Original assignee: United States Biochemical Corporation
Priority date: 1993-02-18
Filing date: 1994-02-16
Publication date: 1994-09-01
Also published as: AU6353194A

Abstract

Method for DNA sequencing comprising the steps of contacting a primer template complex with a T7-type DNA polymerase in the presence of one to three deoxynucleotide triphosphates, wherein one deoxynucleoside triphosphate comprises a biotin moiety, under primer extension conditions.

Description

DESCRIPTION

DNA Sequencing With Non-Radioactive Label

Background of the Invention

This invention relates to methods for DNA sequencing without the use of radioisotopes.

Beck, 164 Analytical Biochemistry 514, 1987, and Beck et al . , 17 Nucleic Acids Research 5115, 1989, describe methods for DNA sequencing using a biotinylated oligonucleotide. The oligonucleotide is labeled with biotin conjugated to its 5' terminus by chemical synthesis, and is used as a primer for dideoxy DNA sequencing in a primer extension reaction. Beck et al. specifically describe use of an enzyme which catalyzes a chemiluminescent reaction in conjunction with biotinlabeled primers.

Pohl and Beck, 155 Methods in Enzvmolocry 250, 1987, describe an apparatus by which DNA sequencing can be more readily performed.

Coull et al., 27 Tetrahedron Letters 3991, 1986, describe a method for synthesis of an oligonucleotide having a 5' primary amino terminus which can be derivatized to give an oligonucleotide biotin adduct.

Agrawal et al. , 14 Nucleic Acids Research 6227, 1986, describe efficient methods for attaching non-radioactive labels to the 5' end of synthetic oligonucleotides. These synthetic oligonucleotides can then be used as primers for DNA sequencing.

Summary of the Invention

This invention features a method and kit for non- isotopic DNA sequencing using biotinylated nucleotides. The invention relates to a method for sequencing DNA without the use of radioisotopes. Following annealing of a sequencing primer to the DNA template to be sequenced, a biotin-labeled nucleoside triphosphate (Bio-dNTP) is incorporated into a DNA chain in an initial labeling reaction catalyzed by a T7-type DNA polymerase (examples of T7-type phage include T7, T3, φl , φll, H, W31, gh-1, Y, A1122, and Sp6) , e.g. , SEQUENASE^® Version 2.0 T7 DNA Polymerase, in which one of the four deoxyribonucleoside triphosphates (dNTPs) is left out of the reaction. In this manner, chain extension is limited (to the point at which the missing dNTP would be incorporated) and a fixed number of biotinylated nucleotides are thereby incorporated. This labeling step is followed by a termination step in which the extended DNA is subdivided into four tubes, each containing all four dNTPs, and one of the four dideoxynucleoside triphosphates (ddNTPs) . DNA polymerase is then added to catalyze further extension and specific termination of the DNA chains. The set of nested fragments produced in each of the four termination tubes can be separated on a denaturing polyacrylamide gel . For example, the fragments are transferred to a nylon membrane by a capillary blotting procedure, and detected using streptavidin-alkaline phosphatase conjugate, and a chemiluminescent substrate for alkaline phosphatase. The light produced is detected on standard X-ray film.

Thus, in a first aspect, the invention features a method for DNA sequencing including the step of providing a DNA primer annealed with a DNA template to be sequenced. This primer-template complex is then incubated in the presence of a T7-type DNA polymerase and at most three deoxynucleoside triphosphates, one of which is labeled with a biotin label. The method then features contacting the resulting primer-template (which is now extended to have at least one biotin-labeled nucleoside triphosphate incorporated) in the presence of sufficient amounts of all four deoxyribonucleoside trisphosphates and a chain terminating agent so that a normal dideoxy sequencing reaction is established. After this reaction, the products of the sequencing procedure are separated and analyzed by standard procedures. In a related aspect the invention features a kit which includes a biotin-labeled nucleoside riphosphate and one or more reagents necessary for dideoxy sequencing, including one or more dideoxynucleoside triphosphates, other deoxynucleoside triphosphates, a T7-type DNA polymerase, and any necessary buffers.

The present invention has advantages over current biotin labeled-primer methods for DNA sequencing. Notably, it does not require that the user synthesize a new biotin-labeled primer for each sequencing reaction in a primer walking strategy. Standard primers prepared in the usual way suffice. The use of only three dNTPs in the labeling step to limit chain extension is important because biotin residues cause aberrant migration of DNA fragments in gels. The more biotin present, the slower the fragment migrates. If chain extension were not limited, the shorter chains would contain fewer biotin residues, while the longer ones would contain more. Thus, the position of the longer fragments would be severely altered and a readable sequencing ladder would not be produced. By limiting chain extensions, each final fragment has the same number of biotin residues and the migration of both short and long fragments is reduced proportionately; a readable sequencing ladder results. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments

Non-isotopic detection of a DNA sequence can be based on detection of one or more biotin residues (which are attached to DNA fragments) by taking advantage of the strong affinity of streptavidin for biotin. One non- isotopic sequencing method, as discussed above, entails initiating DNA synthesis with a 5' -biotinylated primer, separating the DNA fragments on a polyacrylamide gel, and transferring these fragments onto a nylon membrane. The membrane is then incubated, after blocking, with a streptavidin-alkaline phosphatase conjugate. A chemiluminescent alkaline phosphatase substrate (such as Lumi-Phos 530) is added (after appropriate washes) , and the membrane is exposed to standard X-ray film.

As an alternative to chemiluminescence, a chromogenic alkaline phosphatase substrate (such as Visiblot-AP) can be added, causing a dye to be deposited on the membrane where the alkaline phosphatase is bound. The sequence can then be read directly from the membrane.

In the method of this invention, detailed below, biotin is attached to the sequencing products by the incorporation of a biotinylated nucleotide during a labeling step. The sequencing reactions are similar as those used in standard sequencing protocols with one exception: in place of the standard labeling mix (dNTP's minus dATP, with added alpha-³⁵S-dATP) , a custom labeling mix is used which contains a biotinylated deoxynucleotide and which is missing a normal deoxynucleoside corresponding to the labeled one, and at least one of the other unmodified deoxynucleotides. This mixture is designed so that a fixed number of biotinylated deoxynucleotides is incorporated into each fragment (see below) . The biotinylated sequencing products are separated and detected as described above.

Biotin retards the migration of DNA through a gel, so each fragment must contain the same number of biotin residues to avoid multiple banding patterns. The addition of standard termination mixes after the labeling step dilutes out any remaining labeling mix so that any fragments with extra biotinylated nucleotides are spread out randomly, and are not visible in the final sequence.

Example 1: Labeling Mix Design

A 5X labeling mix was prepared containing only 7.5 μM dGTP and 7.5 μM biotinylated dUTP. Extension of the -40

M13 primer (when annealed to an M13 template) in the presence of this mix results in a short product containing the sequence GUUGU. A string of 4 dTTP's terminates the extension, and each fragment contains 3 biotin residues: M13 -40 Primer - 5' - |GTTTTCCCAGTCACGAC| (SEQ. ID. NO: 1)

M13 Template DNA - 3' -CAAAAGGGTCAGTGCTGCAACATTTTGCTGCC (SEQ. ID. NO: 2)

This extension is then resumed by adding the standard dideoxy termination mixes, resulting in production of dideoxy sequencing fragments with 3 biotin residues per fragment. Specific steps are shown below:

Sequencing Reactions

Anneal DNA to Template

M13 DNA (0.2 μg/μl) 5μl M13 -40 Primer (0.5 pm/μl) lμl

5X Sequenase Reaction Buffer 2μl dH₂0 2μl lOμl

Heat to 65°C for two minutes, then cool slowly (in 15-30 min.) to less than 35°C.

Place on ice.

Labeling Step

To annealed DNA, add:

DTT (0.1 M) lμl Labeling Mix (dilute 1:5 in dH₂0) 2μl

Sequenase Enzyme (dilute 1:8) 2μl

15μl

Incubate at room temperature for 2 minutes.

During this step, aliquot 2.5 μl of each of the dideoxy termination mixes (ddG, ddA, ddT, and ddC) into 4 termination tubes. Cap the tubes and prewarm to 37°C.

Termination Reactions

After the two minute labeling incubation above, aliquot 3.5 μl of the labeled DNA mix to each of the prewarmed termination tubes. Incubate the termination tubes at 37°C for 5 minutes. Add 4 μl stop solution to each tube. Place on ice.

Electrophoretic separation, transfer to membrane, and non-isotopic detection of the sequencing products are performed by standard procedures.

Other embodiments are within the following claims.

(1) GENERAL INFORMATION:

(i) APPLICANT: Parke K. Flick

Barry Nash

(ii) TITLE OF INVENTION: DNA SEQUENCING WITH A

NON-RADIOACTIVE LABEL

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Lyon & Lyon

(B) STREET: 611 West Sixth Street

(C) CITY: Los Angeles

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 90017

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44

Mb storage

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: IBM MS-DOS (Version

5.0)

(D) SOFTWARE: WordPerfect (Version

5.1)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

Prior applications total, including application described below: none

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: WARBURG, RICHARD J.

(B) REGISTRATION NUMBER: 32,327

(C) REFERENCE/DOCKET NUMBER: 200/263

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (213) 489-1600

(B) TELEFAX: (213) 955-0440

(C) TELEX: 67-3510 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTTTTCCCAG TCACGAC 17 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) SEQUENCE DESCRIPTION: SEQ ID NO: CAAAAGGGTC AGTGCTGCAA CATTTTGCTG CC 32

Claims

1. Method for DNA sequencing comprising the steps of: contacting a primer template complex with a T7- type DNA polymerase in the presence of one to three deoxynucleotide triphosphates, wherein one said deoxynucleoside triphosphate comprises a biotin moiety, under primer extension conditions.

2. The method of claim 1 wherein said method further comprises the step of contacting said primer template complex after primer extension with four deoxynucleoside triphosphates and at least one chain terminating agent under conditions in which primer extension can occur until said chain terminating agent is incorporated.

3. A kit comprising a biotinylated nucleoside triphosphate and a T7-type DNA polymerase.