GeneRacer™ Advanced RACE Kit
GeneRacer™ Advanced RACE Kit
With the GeneRacer™ Kit you will:
- Obtain the full-length 5' end of rare transcripts present at fewer than 30 copies per cell
- Generate cDNA from transcripts at least 9 kb in length
- Amplify full-length 5' and 3' ends of gene fragments to identify unknown sequence
You can obtain these results with just one round of PCR. Often there’s no need for nested PCR, so you get results on the first try.
How GeneRacer™ Works
The GeneRacer™ Kit ensures that only transcripts containing full-length cDNA ends are amplified. Figure 1 outlines how the GeneRacer™ Kit works. The advanced protocol starts at the RNA level by specifically targeting only 5' capped mRNA. In subsequent steps the cap is removed and replaced with the GeneRacer™ RNA Oligo. During reverse transcription, this RNA oligo sequence is incorporated into the cDNA. Only cDNA that is completely reversed transcribed will contain this known sequence. 5' RACE PCR is then performed using the homologous GeneRacer™ 5' Primer that is specific to the RNA oligo sequence and a gene-specific primer. The result is amplified DNA that contains the full-length 5' cDNA sequence.
- RNA (mRNA or total) is treated with calf intestinal phosphatase (CIP). CIP removes the 5' phosphate from partial transcripts. Without this 5' phosphate, the GeneRacer™ RNA Oligo is prevented from ligating. CIP does not affect the capped mRNA.
- RNA is treated with tobacco acid pyrophosphatase (TAP), which removes the cap from capped mRNA. This exposes the 5' phosphate and permits ligation of the GeneRacer™ RNA Oligo.
- The GeneRacer™ RNA Oligo is ligated to the TAP-treated mRNA with T4 RNA ligase.
- A cDNA template is generated by reverse transcription (RT) using SuperScript® II or AMV-RT and either the GeneRacer™ Oligo dT Primer, a gene-specific primer, or random primers.
- 5' and 3' ends are PCR amplified from these cDNA templates with the appropriate GeneRacer™ Primer and a gene-specific primer. Only cDNA containing the GeneRacer™ sequences will be PCR amplified.
Sensitivity and Length
To demonstrate the ability of the GeneRacer™ Kit to capture the full-length 5' cDNA end, we amplified the 5' ends of genes with known transcriptional start sites. Starting with total RNA and following the GeneRacer™ protocol we were able to amplify both long transcripts (9 kb) and rare messages present at 0.01%, or 30 copies per cell (Figure 2).
Figure 2. Amplification of a Long Transcript & Rare Message
- Three and a half micrograms of HeLa total RNA was treated according to the GeneRacer™ protocol.
- cDNA was generated with the GeneRacer™ Oligo dT Primer and SuperScript® II RT and then PCR amplified with the GeneRacer™ 5' Primer and a gene-specific primer.
- Genes were amplified with one round of a 35-cycle touchdown PCR protocol.
- Fifteen microliters of a 50 µl PCR reaction was run on a 1.2% agarose E-Gel® and visualized under UV light.
|Lane 1:||HPRT (hypoxanthine phosphoribosyl-transferase), 1.3 kb, 30 copies/cell|
|Lane 2:||SMAP (Thyroid hormone receptor coactivating protein), 3.1 kb|
|Lane 3:||Cleavage and polyadenylation specificity factor, 4.5|
|Lane 4:||TFRC (transferrin receptor), 5.0 kb|
|Lane 5:||IGFIIR (Insulin-like growth factor II receptor), 9 kb, 90% GC-rich at the 5' end|
|MW:||100 bp DNA ladder and 1 kb DNA ladder|
In addition to capturing the full-length 5' end of genes with known transcriptional start sites, GeneRacer™ captures additional 5' end sequence of genes with unknown start sites. cDNA amplified with GeneRacer™ was sequenced. Those results were compared to the mRNA sequences listed in GenBank®. GenBank® sequences are typically derived from cDNA obtained from libraries constructed using traditional methods that often result in a loss of terminal nucleotides. As Table 1 clearly shows, the 5' cDNA ends generated with GeneRacer™ extend from 16 to 116 base pairs beyond that listed in GenBank®.
Table 1 - Sequence Beyond Genbank®
|Gene||GenBank® Number (mRNA)||Size (kb)||GeneRacer™ Sequence Beyond GenBank®|
|Heterogeneous nuclear ribonuclear protein complex K protein (hnRNP K)||S74678||2.3||62|
|Subunit for coatomer complex||X70476||3.1||38|
|Muscle phosphofructokinase (PFKM)||M26066||2.8||16|
|ADP-ribosylation factor 4 (ARF4)||M36341.1||1.5||116|
|Putative tumor suppressor (LUCA 15)||U23946.1||2.6||81|
|Thyroid hormone receptor coactivating protein (SMAP)||NM_006696.1||3.1||52|
|Cleavage and polyadenylation specificity factor||U37012.1||4.5||57|
|Human insulin-like growth factor 2 receptor||NM000876||9||44|
Genes listed were amplified using the GeneRacer™ protocol. PCR products were gel purified using S.N.A.P.™ gel purification columns and cloned into the pCR®4-TOPO® vector (Invitrogen). For each gene, 12 clones were randomly picked and sequenced. Sequences were aligned against those in the GenBank® database and sequence beyond the 5' end GenBank sequence identified. The number of additional bases beyond the first base of GenBank sequence is taken from the clone with the longest sequence for each gene.
- Isoleucil-tRNA synthetase had two strong 5' GeneRacer™ PCR products. Sequencing results showed that this gene produced two mRNA populations, one having a deletion, possibly an alternatively spliced intron, at the 5'-end of the mRNA.
Important Licensing Information
"This product is subject to Limited Use Label License Nos. 4, 5, and 54. The Zero Blunt® TOPO Cloning® Kit for Sequencing is sold under a license from Université Libre de Bruxelles."