BDSMARTRACEcDNAAmplificationKitUserManual(English)

SMART

RACE

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BD SMART

™

RACE

cDNA Amplification Kit

User Manual

Cat. No. 634914 or K1811-1

PT3269-1 (PR38330)

Published 09/18/2003

Protocol No. PT3269-1 www.bdbiosciences.com BD Biosciences Clontech

Version No. PR38330 3

BD SMART™ RACE cDNA Amplification Kit User Manual

Table of Contents

continued

List of Figures

Figure 1. Mechanism of BD SMART cDNA synthesis 4

Figure 2. Overview of the BD SMART RACE procedure 6

Figure 3. The relationship of gene-specific primers to the cDNA template 13

Figure 4. 5'- and 3'-RACE sample results 20

Figure 5. Detailed mechanism of the 5'-RACE reactions 37

Figure 6. Detailed mechanism of the 3'-RACE reactions 38

Figure 7. Mechanisms of suppression PCR and step-out PCR 40

List of Tables

Table I: Additional 5'-RACE sequence obtained with

BD SMART technology 5

Table II: Setting up the positive control RACE experiment 19

Table III: Setting up 5'-RACE PCR reactions 21

Table IV: Setting up 3'-RACE PCR reactions 22

Notice to Purchaser

This product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor

is it intended for human use. BD Biosciences Clontech products may not be resold, modified for resale, or used to

manufacture commercial products without written approval of BD Biosciences Clontech.

BD SMART™ technology is covered by U.S. Patent Nos. 5,962,271 & 5,962,272.

Suppression PCR is covered by U.S. Patent No. 5,565,340. Foreign patents pending.

This product is optimized for use in the Polymerase Chain Reaction ("PCR") covered by patents owned by Hoffmann-

La Roche and F. Hoffmann-La Roche, Ltd. ("Roche"). No license under these patents to use the PCR process is

conveyed expressly or by implication to the purchaser by the purchase of this product. A license to use the PCR Process

for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers

such as BD Biosciences Clontech, when used in conjunction with an authorized thermal cycler, or is available from

Perkin-Elmer Corporation. Further information on purchasing licenses to practice the PCR process may be obtained

by contacting the Director of Licensing at the Perkin-Elmer Corporation, 850 Lincoln Centre Drive, Foster City, CA

94404, or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501.

NucleoBond® and NucleoTrap® are registered trademarks of

Macherey-Nagel GmbH & Co. K.G

GeneAmp® is a registered trademark of Roche Molecular Systems, Inc., licensed to The Perkin-Elmer

Corporation.

BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company.

BD Biosciences Clontech www.bdbiosciences.com Protocol No. PT3269-1

4 Version No. PR38330

BD SMART™ RACE cDNA Amplification Kit User Manual

I. Introduction & Protocol Overview

The BD SMART™ RACE cDNA Amplification Kit provides a novel method for

performing both 5'- and 3'-rapid amplification of cDNA ends (RACE). This kit

integrates our BD Marathon™ cDNA Amplification Kit (Chenchik

et al.

, 1995;

1996) with our BD SMART (Switching Mechanism At 5' end of RNA Transcript)

cDNA synthesis technology. This powerful combination allows you to isolate the

complete 5' sequence of your target transcript more consistently than ever

before. Furthermore, BD SMART technology eliminates the need for problematic

adaptor ligation and lets you use first-strand cDNA directly in RACE PCR, a

benefit that makes RACE far less complex and much faster (Chenchik

et al.

1998). The BD SMART RACE Kit also includes recent advances in PCR

technology that both increase the sensitivity and reduce the background of the

RACE reactions. As a result you can use either poly A

or total RNA as starting

material for constructing full-length cDNAs of even very rare transcripts.

BD SMART technology provides a mechanism for generating full-length cDNAs

in reverse transcription reactions (Zhu

et al

., 2001). This is made possible by the

joint action of the BD SMART II™ A Oligonucleotide and the BD PowerScript™

Reverse Transcriptase (RT). BD PowerScript RT is a variant of MMLV RT that,

upon reaching the end of an RNA template, exhibits terminal transferase activity,

adding 3–5 residues (predominantly dC) to the 3' end of the first-strand cDNA

(Figure 1). The BD SMART oligo contains a terminal stretch of G residues that

anneal to the dC-rich cDNA tail and serves as an extended template for RT.

BD PowerScript RT switches templates from the mRNA molecule to the

BD SMART oligo, generating a complete cDNA copy of the original RNA with the

additional BD SMART sequence at the end. Since the dC-tailing activity of RT is

most efficient if the enzyme has reached the end of the RNA template, the

BD SMART sequence is typically added only to complete first-strand cDNAs.

This process guarantees that the use of high quality RNA will result in the

formation of a set of cDNAs that have a maximum amount of 5' sequence

(Table I).

Figure 1. Mechanism of BD SMART™ cDNA

synthesis. First-strand synthesis is primed using a

modified oligo (dT) primer. After reverse transcriptase

reaches the end of the mRNA template, it adds

several dC residues. The BD SMART II A Oligo-

nucleotide anneals to the tail of the cDNA and serves

as an extended template for BD PowerScript RT.

First-strand synthesis

coupled with

(dC) tailing by RT

Poly A

RNA

polyA 3'

BD SMART II

Oligonucleotide

Oligo (dT) primer

Template switching

and extension by RT

polyA

GGG

CCC

Protocol No. PT3269-1 www.bdbiosciences.com BD Biosciences Clontech

Version No. PR38330 5

BD SMART™ RACE cDNA Amplification Kit User Manual

I. Introduction & Protocol Overview

continued

Following reverse transcription, the first-strand cDNA is used directly in 5'- and

3'-RACE PCR reactions, without the need for tedious second-strand synthesis

and adaptor ligation. The incorporation of BD SMART technology also permits

the use of “universal priming” in the RACE PCR amplification. This method,

along with the techniques of suppression PCR and step-out PCR ensure high

specificity in amplifying your target cDNA. These methods are described in detail

below and in Appendix C.

The only requirement for BD SMART RACE cDNA amplification is that you know

at least 23–28 nucleotides (nt) of sequence information in order to design gene-

specific primers (GSPs) for the 5'- and 3'-RACE reactions. (Additional sequence

information will facilitate analysis of your RACE products.) This limited require-

ment makes BD SMART RACE ideal for characterizing genes identified through

diverse methods including cDNA subtraction, differential display, RNA finger-

printing, ESTs, library screening, and more.

BD SMART RACE cDNA amplification is a flexible tool—many researchers use

this kit in place of conventional kits to amplify just the 5' or 3' end of a particular

cDNA. Others perform

both

5'- and 3'-RACE, and many then go on to clone full-

length cDNAs using one of the two methods described in the latter part of this

protocol. In many cases, researchers obtain full-length cDNAs without ever

constructing or screening a cDNA library.

TABLE I: ADDITIONAL 5'-RACE SEQUENCE OBTAINED WITH BD SMART TECHNOLOGY

Additional Matches Includes

Size of sequence genomic transcription

Human gene mRNA (kb) (bp)* sequence start site

Transferrin receptor 5.0 +25 yes yes

Smooth muscle g-actin 1.28 +31 yes yes

Vascular smooth muscle α-actin 1.33 +17 yes yes

Cytoskeletal γ-actin 1.9 +1 yes yes

23 kDa HBP 0.67 +9 n/a yes

p53 2.6 +4 yes yes

Interferon-γ receptor 2.06 +14 yes yes

14-3-3 protein 1.03 +1 n/a n/a

Interferon-α receptor 2.75 +17 yes yes

n/a = not available

* Compared to GenBank cDNA sequence

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BD SMART RACE cDNA Amplification Kit User Manual(English)

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gNDA和cDNA有什么区别？

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