Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-08-07
2002-10-15
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C536S023100, C536S024500
Reexamination Certificate
active
06465219
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and kits for selecting polynucleotide pools from a sample and the selected polynucleotide pools produced thereby. In particular, this invention provides a method for preparing a polynucleotide pool enriched in high-abundance sequences relative to the sample and a subtractive hybridization method for using such a polynucleotide pool to prepare a polynucleotide pool enriched in low-abundance sequences.
BACKGROUND OF THE INVENTION
Biologically active proteins have been the subject of intense research as candidates for therapeutic, diagnostic, and other applications. The first step is these efforts is typically the cloning of the gene encoding the protein from messenger RNA (mRNA). The mRNA of human and other mammalian cells can be divided into three frequency classes: (1) high-abundance sequences, which represent about 10-20% of the total mRNA population; (2) medium-abundance sequences, which represent about 40-45% of mRNA, and (3) low-abundance sequences, which represent another 40-45% of mRNA. Many genes encoding proteins with important regulatory functions, such as hormones and their receptors, are expressed at a low level and the corresponding transcripts fall into the low-abundance class of sequences.
Efforts to clone low-abundance sequences have employed normalized cDNA libraries in which the frequencies of all clones in the library are within a narrow range. However, this approach does not address the loss of low-abundance sequences in the process of generating the cDNA library, which preferentially replicates medium- and high-abundance sequences as well as shorter sequences. A method that facilitated the selection of a pool low-abundance polynucleotides from an mRNA population and that provided a means to produce large amounts of such sequences, without the losses that accompany cloning, would greatly assist research aimed at identifying important regulatory proteins. Ideally, such methods would be capable of replicating a broad range of transcripts without prior cloning into vectors and without requiring knowledge of sequence. Preferably, the low-abundance polynucleotides produced by such methods would be representative of the full-length transcripts (e.g., fill-length cDNA clones).
SUMMARY OF THE INVENTION
The invention provides a method for preparing a selected polynucleotide pool from a polynucleotide sample. In a preferred embodiment, the selected polynucleotide pool is enriched in one or more high-abundance polynucleotides relative to the polynucleotide sample. The method entails synthesizing first antisense polynucleotide strands from sense polynucleotides of, or prepared from, the polynucleotide sample using an antisense primer complex. The antisense primer complex includes an antisense primer operably linked to an RNA promoter sequence, such that the RNA promoter sequence is 5′ of the antisense primer. Next, a universal primer site is added to the 3′ ends of the first antisense polynucleotide strands. The resultant first antisense polynucleotide strands are then diluted to substantially eliminate at least some low-abundance first antisense polynucleotide strands. After dilution, first double-stranded polynucleotides are produced from the remaining first antisense polynucleotide strands. The first double-stranded polynucleotides are enriched in high-abundance polynucleotide sequences relative to the polynucleotide sample.
In a preferred embodiment of the method, the polynucleotide sample is an mRNA sample, the first antisense polynucleotide strands are first antisense cDNA strands, and the first double-stranded polynucleotides are first double-stranded cDNA molecules. The synthesis of first antisense cDNA strands can be primed using a random primer or an oligonucleotide-dT primer. The universal primer site can be added to the 3′ end of the first antisense cDNA strands by template switching, oligonucleotide-tailing, or ligation. The RNA promoter sequence is conveniently one that is recognized by a bacteriophage RNA polymerase, such as T7, T3, or SP6 polymerase.
Preferably, the first double-stranded polynucleotides are produced by amplifying the first antisense polynucleotide strands remaining after dilution, and the amplification is carried out using a universal primer that hybridizes to the universal primer site as the 5′ primer and using the antisense primer complex as the 3′ primer. Most preferably, the amplification is performed by enhanced polymerase chain reaction. This reaction produces a pool of double-stranded polynucleotides that are enriched in high-abundance sequences relative to the original polynucleotide sample. The method optionally includes synthesizing first antisense RNA molecules from the first double-stranded polynucleotides. This pool of antisense RNA molecules is enriched in high-abundance sequences and can therefore be used as a “driver” in subtractive hybridization.
The invention also provides a method of using antisense polynucleotide strands, preferably the high-abundance-enriched antisense RNA molecules prepared as described above, to produce a selected polynucleotide pool from a polynucleotide sample. In a preferred embodiment, the selected polynucleotide pool is enriched in one or more low-abundance polynucleotides relative to the polynucleotide sample. The method entails hybridizing first antisense polynucleotide strands to sense polynucleotide strands of, or prepared from, a polynucleotide sample under hybridization conditions. Preferably, the molar ratio of the first antisense polynucleotide strands to the other polynucleotides in the hybridization mixture is between about 1 and about 100 to 1.
The resulting hybridization mixture includes unhybridized sense polynucleotide strands that are enriched in low-abundance polynucleotide sequences relative to the polynucleotide sample. Second antisense polynucleotide strands are synthesized from the unhybridized sense polynucleotide strands using an antisense primer or an antisense primer complex. The antisense primer complex includes an antisense primer operably linked to an RNA promoter sequence, such that the RNA promoter sequence is 5′ of the antisense primer. An antisense primer complex is preferably employed if it is desirable to produce a pool of selected polynucleotides that each include an RNA promoter to facilitate the synthesis of antisense RNA from the selected polynucleotides.
Next, a universal primer site is added to the 3′ ends of the second antisense polynucleotide strands. Second double-stranded polynucleotides are then produced from the second antisense polynucleotide strands. This pool of polynucleotides is enriched in low-abundance polynucleotide sequences relative to the polynucleotide sample.
In a preferred embodiment of the method, the polynucleotide sample is an mRNA sample, the sense polynucleotide strands are mRNA molecules, the second antisense polynucleotide strands are second antisense cDNA strands, and the second double-stranded polynucleotides are second double-stranded cDNA molecules. The synthesis of second antisense cDNA strands can be primed using an oligonucleotide-dT primer. The universal primer site can be added to the 3′ end of the second antisense cDNA strands by template switching, oligonucleotide-tailing, or ligation. If an antisense primer complex is employed, the RNA promoter sequence is conveniently one that is recognized by a bacteriophage RNA polymerase, such as T7, T3, or SP6 polymerase.
Preferably, the second double-stranded polynucleotides are produced by amplifying the second antisense polynucleotide strands, and the amplification is carried out using a universal primer that hybridizes to the universal primer site as the 5′ primer and using the antisense primer or antisense primer complex as the 3′ primer. Most preferably, the amplification is performed by enhanced polymerase chain reaction. This reaction produces a pool of double-stranded polynucleotides that is enriched in low-abundance sequences relative to the original po
Genemed Biotechnologies, Inc.
Haliday Emily M.
Horlick Kenneth R.
Quine Intellectual Property Law Group P.C.
Strzelecka Teresa
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