Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-07-17
2002-04-30
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S091200, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06379932
ABSTRACT:
FILED OF THE INVENTION
The field of the invention is amplifying RNA.
BACKGROUND
RNA is a frequent starting material for genetic analysis, such as microarray-based diagnostics and sequencing, and a wide variety of methods have been devised to amplify RNA, generally by first copying the RNA to cDNA and then using PCR and/or repeated rounds of transcription to obtaine an amplified product. For example, Silver et al. (1992) U.S. Pat. No. 5,104,792; Liang et al. (1997) U.S. Pat. No. 5,599,672; and Shuber (1999) U.S. Pat. No. 5,882,856 describe methods for amplifying RNA. The present invention provides an improved method of amplifying RNA which is adaptable to total RNA input, low quantity input (100 pg or less mRNA) and linear or quantitative PCR amplification.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for amplifying RNA sequences. In one aspect, the invention comprises the steps of:
(a) hybridizing to a target RNA a first primer comprising a 3′ target RNA hybridizing sequence and a first 5′ defined amplifiable sequence;
(b) extending the first primer with a reverse transcriptase to form a first cDNA strand;
(c) hybridizing to the first cDNA strand a second primer comprising a 3′ random EDNA hybridizing sequence and a second 5′ defined amplifiable sequence;
(d) extending the second primer with a DNA polymerase to form a second cDNA strand; and
(e) amplifying the second cDNA strand with a third primer comprising the first 5′ defined amplifiable sequence.
In one principal embodiment, step (b) yields a heteroduplex of the target RNA and the first cDNA and flier comprises the step of digesting the target RNA of the heteroduplex with a RNase sufficient to permit hybridization of the first cDNA strand with the second primer without a melting step. In various applications, the 3′ target RNA hybridizing sequence may be random or nonrandom, such as complementary to a predetermined sequence (e.g. a coding region, a polyA junction, or a polyA tail), and the first and second 5′ defined amplifiable sequences may be the same or different. In particular embodiments wherein the first and second 5′ defined amplifiable sequences are different, the method further comprises the step of functionally depleting the first primer between steps (b) and (c); step (e) further comprises amplifying the second cDNA strand with a fourth primer comprising the second 5′ defined amplifiable sequence; and/or the method further comprises step (D amplifying the amplified cDNA with an excess of either the third or fourth primer to form a predominantly single stranded amplified probe of a predetermined orientation. In particular applications, the method may be practiced in a single tube (homogeneous assay).
In another principal embodiment, the 3′ target RNA hybridizing sequence is random; the first and second 5′ defined amplifiable sequences are different; and step (b) comprises the step of functionally depleting the first primer to prevent it from hybridizing with the first cDNA strand in subsequent steps.
In aspects of both principal embodiments, interference by the first and/or second primers with the amplification step (e) may be reduced by adding the third and/or fourth primer of step (e) in functional excess of the first and/or second primer; and/or functionally depleting remaining first and second primers between steps (d) and (e).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more and the term “or” means and/or.
The first step of the disclosed methods comprises (a) hybridizing to a target RNA a first primer comprising a 3′ target RNA hybridizing sequence and a first 5′ defined amplifiable sequence. A wide variety of target RNAs may be employed, including total cellular RNA, amplified RNA, purified RNA species such as rRNA, tRNA or preferably, mRNA, etc. The fist primer comprises a 3′ sequence of length and sequence sufficient to hybridize with the target RNA. Depending on the application, this 3′ hybridizing sequence may be random, specific or a combination of random and specific sequences. For example, a primer population comprising random 3′ hybridizing sequences provides a “universal” primer set capable of targeting any RNA species. In other embodiments, primers comprising polyT 3′ hybridizing sequences may be used to target polyA tails of mRNA; primers comprising predetermined specific sequences may be used to target particular, predetermined RNA species comprising complementary sequences; and primers comprising a random region joined to a wobble nucleotide (A, C or G) joined to a polyT region may be used to target mRNA polyA junctions. The first primer also comprises a first 5′ defined amplifiable sequence, which may be any sequences which can be used in the subsequent specific amplification step and preferably comprises a PCRable tag. Suitable reaction conditions for effecting hybridization between the target RNA and first primer are known in the art, readily ascertained empirically, and/or described and/or exemplified herein.
The second step of the methods comprises (b) extending the first primer with a reverse transcriptase to form a first cDNA strand. Depending on the application, an RNAse activity may be present during this step, which can effect the degradation of the original RNA template subsequent to, or coincident with reverse transcription, allowing, for example, priming of the new cDNA strand with the same primer. In a preferred embodiment, the RNAse activity is provided by the reverse transcriptase, such as Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase (both available from Promega, Madison, Wis.), Rous Associated Virus 2 (RAV2) and Human Immunodeficiency Virus 1 (HIV1) reverse transcriptases (both available from Amersham Pharmacia), etc. In applications where the 3′ target RNA hybridizing sequence is random and the first and second 5′ defined amplifyable sequences are different, RNAse activity is preferably avoided so that second strand cDNA synthesis does not occur in the same reaction mixture. Exemplary suitable reverse transcriptases without RNase activity include MMLV-RT RNase H minus (e.g Promega Catalog #M5301 and #M3682), display THERMO-RT (Display Systems Biotech, Vista Calif.), Strat-Script RT (Stratagene, San Diego, Calif.), etc.
The third step of the methods comprises (c) hybridizing to the first cDNA strand a second primer comprising a 3′ random cDNA hybridizing sequence and a second 5′ defined amplifiable sequence. Note that depending on the application, the second primer may have the same 3′ hybridizing sequence and/or the same 5′ defined amplifiable sequences as does the first primer, or one or both sequences may differ; see examples, below. For example, where the first and second 5′ defined amplifiable sequences are different, the method may also comprise the step of functionally depleting the first primer between steps (b) and (c). Functional depletion reduces interference of the first primer with the second primer extension reaction and may be effected by any convenient means such as removal (e.g. size exclusion or affinity chromatography), inactivation (e.g. hydrolysis, conjugation, etc.), etc.
The fourth step of the methods comprises (d) extending the second primer with a DNA polymerase to form a second cDNA strand. Suitable DNA polymerases and reaction conditions are known in the art, readily ascertained empirically, and/or described and/or exemplified below.
The fifth step of the method comprises (e) amplifying the second cDNA strand with a third primer comprising the first 5′ defined amplifiable sequence. To reduce interference from the first pr
Arnold Lyle
Bjeldanes Erik
Daniel Steve
Incyte Genomics Inc.
Lu Frank
Osman Richard Aron
Whisenant Ethan C.
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