Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-05-07
2001-05-08
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C435S091200, C536S023100, C536S024330, C536S025300
Reexamination Certificate
active
06228999
ABSTRACT:
BACKGROUND OF THE INVENTION
It is known in the art of molecular biology that a nucleic acid fragment lying between two identified and unique primer sequences can be amplified using the polymerase chain reaction (PCR) or modifications of the PCR. PCR avoids conventional molecular cloning techniques that require the existence in nucleic acid of advantageous restriction endonuclease cleavage sites. One identified shortcoming of PCR is that fragments greater than about 40 kilobase pairs between the PCR primers are only weakly amplified. It has been difficult to obtain meaningful sequence data from large genomic fragments, particularly when such fragments are resistant to traditional cloning methods. Thus, the art is seeking new methods to obtain the nucleic acid sequences of long, uncharacterized regions of genetic material.
Efforts to amplify a specific DNA cleavage fragment from a population of such fragments have included methods that involve cleaving the DNA using Class IIS enzymes or interrupted palindrome enzymes to form fragments having non-specific terminal 5′ or 3′ overhangs of various lengths (generally 2 to 5 bases). Smith, D. R.,
PCR Methods and Applications
2:21-27, Cold Spring Harbor Laboratory Press (1992); Unrau, P. and K. Deugau,
Gene
145:163-169 (1994); U.S. Pat. No. 5,508,169 (Deugau et al.); Zheleznaya, L. A. et al.,
Biochemistry
(Moscow) 60:1037-1043 (1995). Class IIS enzymes cleave DNA asymmetrically at precise distances from their recognition sequences. Interrupted palindrome (“IP”) enzymes cleave symmetrically between a pair of interrupted palindromic binding sites. To amplify the products of such cleavages, nucleic acid indexing linkers, containing protruding single strands complementary to the cohesive ends of Class IIS- or IP cleavage sites (rather than recognition sequences) and PCR primer sites, have been annealed and ligated to fragments generated by Class IIS- or IP cleavage.
The overhangs vary in base composition, and are determined by the locations of the enzymes' cleavage sites in a genome. The base composition and sequence of the overhang created after cleavage with a Class IIS or IP enzyme cannot be predicted because the sites at which those enzymes cleave DNA are determined by spatial relationship to the recognition sequence, but are not sequence-determined. In the methods described by Smith, Unrau, Deugau and Zheleznaya, the unique cleavage sites generated by Class IIS and IP enzymes determined a random sequence by which fragments could be indexed. However, that is not the case with more popular Class II enzymes that cleave within their recognition sites and generate predictable, identical sticky ends on each restriction fragment. Also, Unrau's method employs temperatures that result in a problem of illegitimate base pairing as well as problems with primer dimers, where indexing fragments anneal with one another rather with the target DNA.
What is desired is an indexing system that relies upon fragments not generated by Class IIS or IP enzymes, and which offer improved amplification specificity.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that oligonucleotide adaptors for directing PCR amplification can be engineered to efficiently and selectively hybridize “fragment indexing sequences” of one or more bases immediately adjacent to a Class II restriction enzyme recognition sites at the termini of a nucleic acid fragment. A Class II enzyme cleaves nucleic acid within its recognition site to generate a characteristic 5′ or 3′ overhanging end or blunt end. The recognition site can include one or more bases that do not form part of the end that results from enzymatic cleavage. When the adaptor and the nucleic acid fragment are brought together under conditions suitable for inter-strand hybridization, the invading strand of the adaptor displaces a portion of the nucleic acid fragment.
Each oligonucleotide adaptor comprises a duplex portion and a single-stranded portion. The duplex portion comprises an invading strand and a complementary PCR primer strand hybridized to the invading (displacing) strand. The oligonucleotide adaptors for the two termini are distinct, in that the PCR primer strands (and their complements on the invading strand) of each end adaptor are selected to specifically amplify fragments in the forward or reverse direction. In the case of adapters that mate with 5′ overhanging termini, the PCR primer strand, which contains the sequence that is the same as that used for a PCR primer, provides a 3′-OH group that is required to join the adaptor to the restriction fragment in the method. The invading strand, which is longer than the PCR primer strand, also includes a protruding single-stranded portion that comprises (1) a nucleic acid sequence that can hybridize to the characteristic overhang and (2) an adaptor indexing sequence that is perfectly complementary to the fragment indexing sequence. The adaptor indexing sequence is provided at the 5′ end of the single-stranded portion of the invading strand. In the case of adapters that mate with 3′ overhanging termini, the primer-complementary strand provides a 5′ phosphate for ligation to the 3′ overhanging end of the restriction fragment.
The invention is further summarized in that oligonucleotide adaptors of the type described can be used in a method for amplifying a restriction fragment that includes the steps of:
(a) cleaving linear or circular nucleic acid at a restriction enzyme recognition site with at least one rare-cutting Class II restriction enzyme to generate a linear restriction fragment having a characteristic 5′ or 3′ overhang at each fragment terminus;
(b) hybridizing to each terminus of the fragment an end-specific oligonucleotide adaptor, thereby displacing one strand of the fragment;
(c) enzymatically ligating the restriction fragment to the primer strand to form a strand-displaced structure; and
(d) amplifying the strand-displaced structure.
The invention is further summarized in that a combinatorial degenerate mixture of oligonucleotide adaptors comprising every indexing sequence is also useful in a method for combinatorial indexing.
In a related aspect, the invention is summarized in that in a method for combinatorial indexing, genetic material cleaved with a rare-cutting enzyme produces a set of fragments for subsequent amplification. The cleaved DNA is added into an array of separate amplification reactions, where each reaction contains both an adaptor specific for one fragment indexing sequence and the degenerate combinatorial mixture of all indexing adaptors specific to the other end of the fragment. Undesired complexity in reaction processing is avoided by including both the single end-specific adaptor and the combinatorial array of adaptors in the hybridization step.
In addition to obtaining valuable sequence data from the amplified fragments, it is possible to order the fragments by generating a restriction map by performing cross-digestion using two or more different enzyme arrays. By selecting the adaptor sequence, various PCR-related methods can be employed directly on the amplification products, including PCR sequencing.
It is an object of the present invention to facilitate accessing and sequencing regions of the human genome that are resistant to molecular cloning.
It is another object of the present invention to amplify nucleic acid fragments with specificity.
It is a feature of the present invention that the overhang generated by cleavage with a Class II enzyme is predictable and invariant for each enzyme.
It is another feature of the present invention that the indexing sequence is separate from (not a part of) the overhang generated by restriction enzyme cleavage.
It is yet another feature of the present invention that a degenerate collection of adaptors containing all possible indexing sequences is used in combination with a defined adaptor duplex to amplify unknown sequences of enzyme-cleaved nucleic acid.
It is an advantage of the pres
Guilfoyle Richard A.
Guo Zhen
Marschel Ardin H.
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
LandOfFree
Nucleic acid indexing does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid indexing, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid indexing will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2473603