Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-14
2002-05-14
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100, C536S024300
Reexamination Certificate
active
06387624
ABSTRACT:
FIELD OF INVENTION
The present invention relates generally to the field of amplifying nucleic acids, more particularly to methods for producing cDNA from mRNA, sequencing DNA, and constructing cDNA libraries.
BACKGROUND OF THE INVENTION
The characterization of cell specific gene expression finds application in a variety of disciplines, such as in the analysis of differential expression between different tissue types, different stages of cellular growth or between normal and diseased states, and the like. Fundamental to the characterization of cell specific gene expression is the detection of mRNA, and the construction of comprehensive cDNA libraries. However, the detection of mRNA is often complicated by one or more of the following factors: cell heterogeneity, paucity of material, or limits of low abundance mRNA detection.
In a general method of constructing cDNA libraries, polyA mRNA is prepared from the desired cells and the first strand of the cDNA is prepared from the polyA mRNA using a RNA-dependent DNA polymerase (“reverse transcriptase”) and an oligodeoxynucleotide primer of 12 to 18 thymidine residues. In another method, the primer contains one or two nucleotides at one end that can hybridize to the mRNA sequence upstream of the polyA tail. Usually, the first polyA-non-complementary nucleotide is a deoxyadenylate, deoxyguanylate, or deoxycytidylate (“dC”), and the second nucleotide can be any deoxynucleotide. The use of 2 nucleotides can provide a moreaccurate positioning of the primer at the junction between mRNA and the polyA tail.
The second strand of the cDNA is synthesized by one of several methods, the more efficient of which are commonly known as “replacement synthesis” and “primed synthesis.” Replacement synthesis involves the use of ribonuclease H (“RNAase H”), which cleaves the phosphodiester backbone of RNA that is in a RNA:DNA hybrid leaving a 3′ hydroxyl and a 5′ phosphate, to produce nicks and gaps in the mRNA strand, creating a series of RNA primers that are used by
E. coli
DNA polymerase I, or its “Klenow” fragment, to synthesize the second strand of the cDNA. This reaction is very efficient; however, the cDNAs produced most often lack the 5′ terminus of the mRNA sequence.
Primed synthesis to generate the second cDNA strand is a general name for several methods which are more difficult than replacement synthesis yet clone the 5′ terminal sequences with high efficiency. In general, after the synthesis of the first cDNA strand, the 3′ end of the cDNA strand is extended with terminal transferase, an enzyme which adds a homopolymeric “tail” of deoxynucleotides, most commonly deoxycytidylate. This tail is then hybridized to a primer of oligodeoxyguanidylate or a synthetic fragment of DNA with an deoxyguanidylate tail and the second strand of the cDNA is synthesized using a DNA-dependent DNA polymerase.
Once both cDNA strands have been synthesized, the cDNA library is constructed by cloning the cDNAs into an appropriate plasmid or viral vector. In practice this can be done by directly ligating the blunt ends of the cDNAs into a vector which has been digested by a restriction endonuclease to produce blunt ends. Blunt end ligations are very inefficient, however, and this is not a common method of choice. A generally used method involves adding synthetic linkers or adapters containing restriction endonuclease recognition sequences to the ends of the cDNAs. The cDNAs can then be cloned into the desired vector at a greater efficiency.
One potential problem with the current method of constructing cDNA libraries is that the hybridization of the oligo dT primer to the polyA tail of the mRNA in the initial step is not perfect. The primer does not necessarily accurately position at the junction between the mRNA and its polyA tail. Therefore, there may be continuous stretches of T's in addition to the T's on the first strand primer. While this does not usually affect efficiencies in sequencing from the 5′ end, it severly compromises the ability to obtain accurate and successful sequencing from the 3′ (polyA tail) end. Thus, there exists a need for methods and procedures of cDNA synthesis and cloning.
SUMMARY OF THE INVENTION
Methods are provided for obtaining a DNA complementary to a mRNA by contacting the mRNA having a polyadenosine (polyA) tail with a primer mixture, where each primer in the mixture comprises at least 5 contiguous deoxythymidines and at least 2 independently selected non-deoxythymidine nucleotides near one end, and reverse transcribing the mRNA using a reverse transcriptase to produce a DNA strand complementary to the mRNA.
Methods are also provided for obtaining a DNA complementary to a mRNA by contacting the mRNA having a polyA tail with a primer mixture, where each primer in the mixture comprises at least 10 contiguous deoxythymidines and a non-polyA-complementary region near one end, and reverse transcribing the mRNA using a reverse transcriptase to produce a DNA strand complementary to the mRNA. The non-polyA-complementary region is selected from the group consisting of 3′-VV, 3′-VTV, 3′-VTVV, 3′-VTVVV, 3′-VTVVTV, 3′-VTTV, 3′-VTTTV, 3′-VVTVVV, and 3′-VVVVV, and combinations thereof, wherein V is deoxyadenosine, deoxycytidine, or deoxyguanosine, and the primer mixture may contain primers that are sense, anti-sense, or double stranded, and may contain a double stranded restriction enzyme sequence near the end opposite to the one containing the non-deoxythymidine nucleotides.
Methods are also provided for producing uni-directionally cloned complimentary DNA libraries from mRNA by contacting the mRNA having polyadenylated tails with a primer mixture, wherein each primer in the mixture has at least 10 contiguous deoxythymidines and at least two non-deoxythymidine nucleotides near one end and a double stranded restriction enzyme sequence at the opposite end, reverse transcribing the mRNA using a reverse transcriptase to produce a DNA strand complementary to the mRNA, modifying the complementary DNA strand wherein the polyT tail is substantially removed, and amplifying the modified cDNA strand by inserting the strand into a cloning vector uni-directionally, and amplifying using a DNA polymerase.
Methods are also provided for producing uni-directionally cloned complimentary DNA libraries from mRNA by contacting the mRNA having a polyA tail with a primer mixture wherein each primer in the mixture has at least 15 contiguous deoxythymidines having a restriction enzyme site at one end and a non-polyA-complementary region near the opposite end, wherein the non-polyA-complementary region is selected from the group consisting of 3′-VV, 3′-VTV, 3′-VTVV, 3′-VTVVV, 3′-VTVVTV, 3′-VTTV, 3′-VTTTV, 3′-VVTVVV, and 3′-VVVVV, and combinations thereof, wherein V is deoxyadenosine, deoxycytidine, or deoxyguanosine, reverse transcribing the mRNA using a reverse transcriptase to produce a cDNA strand having a polyT tail, modifying the cDNA strand wherein the polyT tail is substantially removed, and amplifying the modified cDNA strand by inserting the strand into cloning vector uni-directionally, and amplifying using a DNA polymerase. The primer mixture may contain primers that are sense, anti-sense, or double stranded, and may contain a restriction enzyme site near the end opposite to the one containing the non-deoxythymidine nucleotides.
These and other objections, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.
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Thweatt et al., Analytical Biochemistry 190:314-316 (1990).*
Moore et al., Nucleic Acids Research 18 (7):1921 (199
Fu Glenn K.
Starnes Steven
Stuve Laura L.
Bozicevic Field and Francis LLP
Francis Carol L.
Incyte Pharmaceuticals Inc.
Whisenant Ethan C.
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