Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-05
2001-01-16
Schwartzman, Robert A. (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024200
Reexamination Certificate
active
06175002
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to improved methods and compositions for analyzing nucleic acids, and more particularly, to a method of analyzing terminal nucleotides of polynucleotides by specific ligation of labeled adaptors.
BACKGROUND
The desire to understand the genetic basis of disease and a host of other physiological states associated different patterns of gene expression has led to the development of several approaches to large-scale analysis of DNA, Adams et al, Editors, Adams DNA Sequencing and Analysis (Academic Press, New York, 1994). Current techniques for analyzing gene expression patterns include large-scale sequencing, differential display, indexing schemes, subtraction hybridization, hybridization with solid phase arrays of cDNAs or oligonucleotides, and numerous DNA fingerprinting techniques, e.g. Lingo et al, Science, 257: 967-971 (1992); Erlander et al, International patent application PCT/US94/13041; McClelland et al, U.S. Pat. No. 5,437,975; Unrau et al. Gene, 145: 163-169 (1994); Schena et al, Science, 270: 467-469 (1995); Velculescu et al. Science, 270: 484-486 (1995); and the like.
An important subclass of such techniques employs double stranded oligonucleotide adaptors to classify populations of polynucleotides and/or to identify nucleotides at the termini of polynucleotides, e.g. Unrau et al (cited above) and U.S. Pat. No. 5,508,169; Sibson, International applications PCT/GB93/01452 and PCT/GB95/00109; Cantor, U.S. Pat. No. 5,503,980; and Brerner, International application PCT/US95/03678 and U.S. Pat. No. 5,552,278. Such adaptors typically have protruding strands which permit specific hybridization and ligation to polynucleotides having complementary ends. Identification or classification is effected by carrying out such reactions in separate vessels or by providing labels which identify one or more nucleotides in the protruding strand of the ligated adaptor.
In these techniques, special problems arise in dealing with either polynucleotide ends or adaptors that are capable of self-ligation, such as that illustrated in
FIG. 1
, where the four-nucleotide protruding strands of the anchored polynucleotides are complementary to one another. When self-ligation occurs, the protruding strands of either the adaptors or the target polynucleotides are no longer available for analysis or processing. This, in turn, leads to the loss or disappearance of signals generated in response to correct ligations of adaptors to target polynucleotides. The self-ligation problem is especially acute when identical target polynucleotides are anchored to a solid phase support. In this situation, the local concentration of ends capable of self-ligation is typically very high relative to that of double stranded adaptors, thereby making self-ligation the favored reaction, whenever complementary sequences are present. As illustrated in
FIG. 1
, complementary sequences form a palindromic duplex upon hybridization. Since the probability of a palindromic 4-mer occurring in a random sequence is the same as the probability of a repeated pair of nucleotides (6.25%), adaptor-based methods for de novo sequencing have a high expectation of failure after a few cycles because of self-ligation. When this occurs, further analysis of the polynucleotide becomes impossible.
In view of the increasing importance of adaptor-based techniques in nucleic acid sequence analysis, the availability of methods and materials for overcoming the self-ligation problem would be highly desirable.
SUMMARY OF THE INVENTION
Accordingly, an object of our invention is to provide an adaptor-based analysis method in which neither the adaptors nor target polynucleotides self-ligate.
Another object of our invention is to provide an improved method of DNA sequencing by ligation wherein identical target polynucleotides anchored to a solid phase support cannot ligate to one another in the presence of a ligase.
A further object of our invention is to provide oligonucleotide adaptors which cannot ligate to one another, but at the same time are capable of being ligated to a target polynucleotide having a complementary protruding strand.
Another object of our invention is to provide compositions comprising uniform populations of identical polynucleotides anchored by one end to a solid phase support, the polynucleotides having at their free ends a protruding strand lacking a 5′ phosphate group.
The invention achieves these and other objectives by providing methods and compositions for analyzing polynucleotides using double stranded adaptors. An important aspect of the invention is the removal of the 5′ phosphate from the end of the polynucleotide to be analyzed so that self-ligation cannot occur, particularly in embodiments employing enzymatic ligation. Another important aspect of the invention is the use of double stranded adaptors each having a protruding strand complementary to that of the polynucleotides to be analyzed and each having a strand with a blocked 3′ carbon so that the strand cannot be ligated. Preferably, double stranded adaptors of the invention are defined by the formulas:
5
′
-
p
(
N
)
n
(
N
)
q
-
3
′
z
(
N
′
)
r
-
5
′
or
p
(
N
)
q
-
3
′
3
′
-
z
(
N
)
n
(
N
′
)
r
-
5
′
where N is a nucleotide and N′ is its complement, p is a phosphate group, z is a 3′ blocking group, n is an integer between 2 and 6, inclusive, q is an integer greater than or equal to 8, and r is an integer greater than or equal to 8, which may be the same or different from q.
Preferably, z is a phosphate group and the double stranded portion of the adaptors contain a nuclease recognition site of a nuclease whose recognition site is separate from its cleavage site. As described more fully below, the latter element is useful in embodiments employing repeated cycles of ligation and cleavage for DNA sequencing.
In a preferred embodiment, illustrated in FIG.
2
a
, the invention provides a method for determining the identify of nucleotides at the terminus of a polynucleotide. The method comprises the following steps: (a) ligating a double stranded adaptor to an end of the polynucleotide, the end of the polynucleotide having a dephosphorylated 5′ hydroxyl and the double stranded adaptor having a first strand and a second strand, the second strand of the double stranded adaptor having a 3′ blocking group; (b) removing the 3′ blocking group after ligation of the first strand; (c) phosphorylating the 5′ hydroxyl of the polynucleotide; (d) ligating a second strand having an unblocked 3′ moiety to regenerate the double stranded adaptor; and (e) identifying one or more nucleotides at the end of the polynucleotide by the identity of the adaptor ligated thereto. The step of removing the 3′ blocking group may be carried out in several ways, including physically removing, e.g. by washing, the entire 3′ blocked second strand followed by replacement with an unblocked strand, by chemically or enzyrmatically removing the 3′ blocking group, or by otherwise activating or regenerating a 3′ hydroxyl on the second strand without removal of the second strand. A 3′ phosphate is the preferred 3′ blocking group and the step of removing is preferably carried out by treating the second strand with a phosphatase to remove the 3′ phosphate and to regenerate a free 3′ hydroxyl.
In further preference, the double stranded adaptor includes a recognition site of a nuclease whose recognition site is separate from its cleavage site. This permits one to carry out repeated cycles of ligation and cleavage to identify additional nucleotides of the target polynucleotide. Accordingly, the method of the invention preferably includes the further steps of (f) cleaving said polynucleotide with a nuclease recognizing said nuclease recognition site of said double stranded adaptor such that said polynucleot
Albrecht Glenn
Brenner Sydney
Dubridge Robert B.
Gryaznov Sergei M.
McCurdy Sarah N.
Gorthey LeeAnn
Larson Thomas G.
Lynx Therapeutics, Inc.
Macevicz Stephen C.
Powers Vincent M.
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