SMARTer_RACE_5_3__Kit_User_Manual_022714(5)

clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual

The SMARTer II A Oligonucleotide contains a terminal stretch of modified bases that anneal to the extended

cDNA tail, allowing the oligo to serve as a template for the RT. SMARTScribe RT switches templates from the mRNA molecule to the SMARTer oligo, generating a complete cDNA copy of the original RNA with the

additional SMARTer sequence at the end. Since the template switching activity of the RT occurs only when the enzyme reaches the end of the RNA template, the SMARTer sequence is typically only incorporated into full-

length, 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).

Table 1. Additional 5'-RACE Sequence Obtained with SMART Technology

Human gene

Piccolo presynaptic cytomatrix protein

Dynein, cytoplasmic 1, heavy chain 1

Polycystic kidney disease 1

Solute carrier family 1

Microtubule-associated protein 1A

Spectrin, beta, non-erythrocytic

Transferrin receptor

Interferon-α receptor

Smooth muscle g-actin Size of mRNA (kb) 20.29 14.36 14.14 12.02 10.54 10.24 5.0 2.75 1.28 Additional sequence (bp)* +59 +36 +21 +73 +13 +32 +25 +17 +31 Matches genomic sequences yes yes yes yes yes yes yes yes yes

Following reverse transcription, SMART technology allows first-strand cDNA to be used directly in 5’- and

3’-RACE PCR reactions. Incorporation of universal primer binding sites in a single-step during first-strand cDNA synthesis eliminates the need for tedious second-strand synthesis and adaptor ligation. This simple and highly

efficient SMARTer cDNA synthesis method ensures higher specificity in amplifying your target cDNA.

Suppression PCR & step-out PCR techniques are used in combination with SMARTer technology to decrease

background amplification in RACE PCR.

Requirements for SMARTer RACE cDNA Amplification

The only requirement for SMARTer 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 requirement

makes SMARTer RACE ideal for characterizing genes identified through diverse methods, including cDNA

subtraction, differential display, RNA fingerprinting, ESTs, library screening, and more.

Uses of SMARTer RACE cDNA Amplification

SMARTer 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.

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