5'RACE LaNe Characterization of Murine
GAPDH
39
M
OLECULAR
B
IOTECHNOLOGY
Volume 29, 2005
RESEARCH
39
Molecular Biotechnology
2005 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2005/29:1/39–46/$30.00
*Author to whom all correspondence and reprint requests should be addressed. *Department of Zoology, The University of Melbourne,
22 Rutland Street, Clifton Hill, Melbourne, Vic., 3068, Australia. E-mail: d.park@unimelb.edu.au.
Abstract
A New 5' Terminal Murine
GAPDH
Exon Identified
Using 5'RACE LaNe
Daniel Jonathan Park*
In this work, a ligation-independent, fully gene-specific, nested polymerase chain reaction (PCR) method
for the elucidation of 5' cDNA sequence is described and demonstrated for the first time. Two manifestations
of the method, rapid amplification of cDNA ends (RACE) by lariat-dependent nested PCR 5' (RACE LaNe),
at least as simple to perform as conventional RACE, were successfully applied to the murine housekeeping
genes phosphoglycerate kinase 1 (PGK1), β-actin (β-ACT), and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and the alpha thalassemia mental retardation Y homolog (ATRY) gene of the marsupial, Macropus
eugenii. Significantly, a new murine GAPDH 5' exon, separated by 365 kb of intronic sequence from previ-
ously annotated GAPDH sequence, was discovered using 5'RACE LaNe.
Index Entries: 5'RACE; RACE LaNe; GAPDH; cDNA; sequencing.
1. Introduction
The 5' rapid amplification of cDNA ends
(5'RACE) (1,2), inverse polymerase chain reac-
tion (PCR) (3), RNA ligase-mediated 5'RACE
(RLM-RACE) (4,5), and CapSelect (6) represent
useful PCR-based approaches for the determina-
tion of 5' flanking cDNA sequence, without the
requirement for library construction. However,
they depend on “one-sided” PCRs or require effi-
cient ligation reactions. The 3'RACE by lariat-
dependent nested PCR (3'RACE LaNe) (7,8) is a
simple and rapid method that allows “two-sided”
gene-specific, fully nested PCR series for the elu-
cidation of 3' flanking cDNA sequence without the
requirement for ligation. Here, two methods for
the application of the 3'RACE LaNe principle to the
context of 5' flanking cDNA sequence (5'RACE
LaNe), are described. First-strand intrastrand-
annealing 5'RACE LaNe (FI RACE LaNe) ex-
ploits the inherent ability of the 3' terminus of
single-stranded cDNA to hybridize intrastrand to
prime second strand synthesis (Fig. 1) (9–12). Ter-
minal transferase-mediated 5'RACE LaNe (TT
RACE LaNe) combines design features of Cap-
Select and RACE LaNe (Fig. 1). FI RACE LaNe
and TT RACE LaNe were applied to the murine
housekeeping genes: phosphoglycerate kinase 1
(PGK1), β-actin (β-ACT), and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) and the α
thalassemia mental retardation Y homolog (ATRY)
gene of the marsupial, the tammar wallaby
(Macropus eugenii).
Significantly, application of FI RACE LaNe
led to the discovery of a new exon for the GAPDH
housekeeping gene, separated from previously
characterized GAPDH cDNA by an intron span-
ning 365 kb. The new splice-form of GAPDH was
subsequently demonstrated to be present in a
range of murine tissues, with implications for tis-
sue-specific gene expression.