Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-15
2001-07-03
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091100, C435S091200, C435S005000, C435S006120, C536S023100, C536S024300, C536S024320, C536S024330, C536S024500
Reexamination Certificate
active
06255082
ABSTRACT:
BACKGROUND OF THE INVENTION
The disclosed invention is generally in the field of nucleic acid amplification.
DNA molecular cloning is routinely carried out using plasmid, phage, or viral vectors that replicate inside cells. Genomic cloning is routinely carried out using vectors that replicate inside cells. While existing cloning methods work quite well for most genomic fragments, certain DNA domains tend to suffer alterations, notably deletions or rearrangements. A method, in which individual DNA molecules are cloned in solution by serial dilution and subsequent PCR amplification from tubes containing single molecules has been described (Lukyanov et al.,
Nucleic Acid Research
24:2194-2195 (1996)). A method has also been described for cloning RNA populations derived from single RNA molecules in an immobilized medium (Chetverina and Chetverin,
Nucleic Acids Research
21:2349-2353 (1993)). While both of these methods allow in vitro cloning, neither is practical for cloning of large fragments.
A number of methods have been developed for exponential amplification of nucleic acids. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q&bgr; replicase (Birkenmeyer and Mushahwar,
J. Virological Methods
, 35:117-126 (1991); Landegren,
Trends Genetics
9:199-202 (1993)).
Current methods of PCR amplification involve the use of two primers which hybridize to the regions flanking a nucleic acid sequence of interest such that DNA replication initiated at the primers will replicate the nucleic acid sequence of interest. By separating the replicated strands from the template strand with a denaturation step, another round of replication using the same primers can lead to geometric amplification of the nucleic acid sequence of interest. PCR amplification has the disadvantage that the amplification reaction cannot proceed continuously and must be carried out by subjecting the nucleic acid sample to multiple cycles in a series of reaction conditions. PCR also has the disadvantage that the length of nucleic acid that can be effectively amplified is limited.
Accordingly, there is a need for a cloning and amplification method that allows amplification of longer nucleic acid segments and that is less complicated, are more reliable, and produces greater amplification in a shorter time.
It is therefore an object of the disclosed invention to provide an in vitro method of cloning and amplifying a target nucleic acid sequence in a continuous, isothermal reaction.
It is another object of the disclosed invention to provide an in vitro method of cloning and amplifying a target nucleic acid sequence where multiple copies of the target nucleic acid sequence are produced in a single amplification cycle.
It is another object of the disclosed invention to provide a kit for cloning and amplifying a target nucleic acid sequence in a continuous, isothermal reaction.
BRIEF SUMMARY OF THE INVENTION
Disclosed are compositions and an in vitro method for cloning and/or amplification of nucleic acid sequences of interest. The method is based on strand displacement replication of the nucleic acid sequences by multiple priming on artificial long terminal repeat (ALTR) sequences appended to the ends of the nucleic acid molecule of interest. The target sequences for cloning and amplification can be very long, up to 40 to 80 Kb or longer. In a preferred form of the method, a single primer is used to prime strand displacement replication at multiple sites in artificial long terminal repeat sequences, flanking a target nucleic acid, containing multiple tandem repeats of a primer complement sequence. Amplification proceeds by replication initiated at each primer and continuing through the target nucleic acid sequence.
A key feature of this method is the displacement of intervening primers during replication. Once the nucleic acid strands elongated from the right ALTR reaches the left ALTR, and vice versa, another round of priming and replication will take place. This allows multiple copies of a nested set of the target nucleic acid sequence to be synthesized in a short period of time. This nested replication of multiple copies significantly increases the amplification yield for extremely long target sequences since copies of the target sequence are produced simultaneously, not sequentially as in PCR. By using a sufficient number of repeat units in the ALTRs, and thus a sufficient number of primer complement sequences, only a few rounds of replication are required to produce hundreds of thousands of copies of the nucleic acid sequence of interest.
The disclosed method has advantages over the polymerase chain reaction since it can be carried out under isothermal conditions. No thermal cycling is needed because the polymerase at the head of an elongating strand (or a compatible strand-displacement protein) will displace, and thereby make available for hybridization, the strand ahead of it. Other advantages of the disclosed method include the ability to amplify very long nucleic acid segments (on the order of 80 kilobases) and rapid amplification of shorter segments (10 kilobases or less). In the disclosed method, single priming events at unintended sites will not lead to artifactual amplification at these sites (since amplification at the intended site will quickly outstrip the single strand replication at the unintended site).
A preferred form of the disclosed method makes use of indexed artificial long terminal repeats that allow amplification and identification of specific nucleic acid fragments present in a mixture of nucleic acid fragments without requiring any knowledge of the sequence of the nucleic acid fragment. This is accomplished by digesting a nucleic acid sample, such a genomic DNA, with a restriction enzyme having an interrupted palindrome recognition sequence or a cleavage site offset from the recognition site (interrupted palindrome and class-IIS restriction enzymes). The resulting fragments having a random distribution of sticky ends are then ligated to pairs of indexed ALTRs in separate reactions collectively representing every combination of indexed ALTRs. The indexed ALTRs each have a different sticky end and the set of ALTRs collectively include all of the possible sticky ends that can be generated by the restriction enzyme used. Thus, in each ligation reaction, only those nucleic acid fragments having sticky ends complementary to the sticky ends of the specific pair of ALTRs present in the reaction will have an ALTR added to each end. As a result, only these fragments to which ALTRs have been added will be amplified. Alternatively, all of the ALTRs can be used together in the same reaction to clone and amplify all of the compatible fragments in a nucleic acid sample. In the resulting mixture of clones, a clone of interest, if identified, can be separately amplified by identifying the ALTRs flanking the clone and using the same ALTRs to clone the fragment individually. Once identified, the same nucleic acid fragment can be cloned from the same source using the same pair of ALTRs that resulting in the original amplification.
Following amplification, the amplified sequences can be for any purpose, such as uses known and established for PCR amplified sequences. For example, amplified sequences can be detected using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. The amplified sequences can also be used to long nucleic acids for pharmaceutical uses, such as for multi-antigen vaccines, that are free of contaminating proteins and other cellular components that are present in nucleic acid replicated in cells.
A key feature of the disclosed method is that amplification takes place not in cycles, but in a continuous, isothermal replication. This makes amplification less compli
Jones W. Gary
Needle & Rosenberg P.C.
Taylor Janell E.
Yale University
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