John M. S. Bartlett.pdf - Bio-Nica.info

Rapid Amplification of cDNA Ends 113
precautions for handling RNA should be followed carefully (10). TRIzol Reagent
(Invitrogen) performs well with small quantities of tissue. Total RNA has been successfully
extracted from punches of rat supraoptic and paraventricular nuclei and used to clone a
gene preferentially expressed in the supraoptic and paraventricular nuclei of the brain
by DD-PCR (unpublished data). High-quality poly (A)+ mRNA can be obtained from
companies such as Clontech Laboratories, Inc.
2. Reverse transcriptase and related buffers supplied in the Marathon cDNA amplification
(Clontech) may be used for the first-strand synthesis to meet most full-length cDNA
cloning needs. SuperScript II (RNAse H-) reverse transcriptase (Invitrogen) is a preferred
reverse transcriptase to generate longer first-strand cDNAs.
3. The oligo-dT primer (5′-TTCTAGAATTCAGCGGCCGC(T) 30 MN-3′) supplied in the
Marathon cDNA amplification kit (Clontech) is suitable as a universal cDNA synthesis primer.
The two degenerate nucleotides (where M=A, G or C; N=A, G, T or C) place the oligo-dT
primer at the beginning of the poly(A) tail and thus eliminate 3′-heterogeneity (11).
4. Combine 25 µL of E. coli DNA polymerase I (10.0 units/µL, Invitrogen), 3 µL of E. coli
DNA ligase (10.0 units/µL, Invitrogen), 3 µL of E. coli RNase H (2.1 units/µL, Invitrogen),
and 10 µL of high-quality H 2 O to make 20× second-strand enzyme mixture. 5× secondstrand
buffer contains 500 mM KCL, 50 mM ammonium sulfate, 25 mM MgCL 2 , 0.75 mM
beta-NAD, 100 mM Tris (pH 7.5), and 0.25 mg/mL bovine serum albumin (Clontech’s
Marathon cDNA Amplification Kit).
5. The primer’s melting temperature is an important parameter that greatly influences PCR
specificity by reducing nonspecific priming events. High-melting point primers with
melting temperatures between 65 and 70°C allow the use of higher annealing temperatures
to enhance reaction specificity. The estimated melting temperature [(G+C) × 4 + (A+T) × 2]
are not exact under PCR conditions but can be used as a starting point. Sense primers are
used for 3′-RACE whereas anti-sense primers are used for 5′-RACE.
6. Touchdown PCR may help eliminate extraneous bands and increase yield. The first round
of touchdown PCR has an annealing temperature 5 to 10°C higher than what is usually
used. In each subsequent round, the annealing temperature is dropped a degree until the
standard annealing temperature is reached.
7. GeneAmp PCR System 9600 (Applied Biosystems) gives more satisfactory amplification
efficiency, especially for low abundant genes. Applied Biosystems GeneAmp 0.5-ml PCR
tubes are preferred PCR tubes for critical PCR amplification.
8. Northern analysis is the most preferred method to provide an expression profile and
size estimation of the mRNA. If northern data is not readily available, a quick reversetranscription
PCR survey using two GSPs (to produce a sizable PCR fragment) could be
used to identify the best tissue source for the actual RACE PCR.
9. The 5′-RACE outlined in Subheading 3.2. is essentially as described in the Marathon
cDNA amplification kit (Clontech Laboratories, Inc.) and the Expand PCR system
(Boehringer Mannheim Biochemicals) except an oligo-dT primer (5′- CACTATAGGCCATC
GAGGCC(T) 20 MN-3′) was used for first-strand cDNA synthesis. The adaptor-ligated
cDNA pool is essentially an uncloned cDNA library, which can be used to isolate fulllength
cDNAs for many different genes using gene-specific primers. These two-sided
(5′ and 3′) anchored cDNAs permit 5′- and 3′-RACE PCR to be performed with the same
cDNA pool. If the known sequence information is far upstream from the 3′ end of the
mRNA (poly A tail), especially for the large transcript of the gene, the 3′-RACE protocol
described in Subheading 3.3. is a preferred strategy to obtain this potentially long (often
untranslated) region quickly and reliably. Although the ligation of the double-stranded
adaptor using T4 DNA ligase is much more efficient compared to the inefficient tailing or
the single-stranded anchor ligation, the adaptor actually tags only a portion of cDNAs. The