Rapid Amplification of cDNA Ends (RACE) is a procedure for amplification of nucleic acid
sequences from a messenger RNA template between a defined internal site and either the 3´
or the 5´ end of the mRNA (1) This methodology of amplification with singlesided specificity
has been described as onesided PCR (2) or anchored PCR (3) PCR requires two
sequencespecific primers that flank the sequence to be amplified (45) However to amplify
and characterize regions of unknown sequences this requirement imposes a limitation (3) 3´
RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for
PCR In this procedure mRNAs are converted into cDNA using reverse transcriptase (RT) and
an oligodT adapter primer Specific cDNA is then amplified by PCR using a genespecific
primer (GSP) that anneals to a region of known exon sequences and an adapter primer that
targets the poly(A) tail region This permits the capture of unknown 3´mRNA sequences that
lie between the exon and the poly(A) tail 5´ RACE uses an antisense gene specific primer for
the synthesis of specific cDNA by reverse transcriptase
Prior to PCR a TdTtailing step attaches an adapter sequence to the unknown 5´ sequences
of the cDNA Specific cDNA is then amplified by PCR using a GSP that anneals in a region of
known exon sequences and an adapter primer that targets the 5´ terminus RACE has been
used for amplification and cloning of rare mRNAs (6) and may be applied to existing cDNA
libraries (7) Additionally RACE products can be directly sequenced without any intermediate
cloning steps (89) or the products may be used to prepare probes (10) Products generated
by the 3´ and 5´ RACE procedures may be combined to generate fulllength cDNAs (1011)
Lastly the RACE procedures may be utilized in conjunction with exontrapping methods (12) to
enable amplification and subsequent characterization of unknown coding sequences
The 3´ RACE procedure is summarized as follows First strand cDNA synthesis is initiated at
the poly(A) tail of mRNA using the adapter primer (AP) After first strand cDNA synthesis the
original mRNA template is destroyed with RNase H which is specific for RNADNA
heteroduplex molecules Amplification is performed without intermediate organic extractions or
ethanol precipitations using two primers one is a userdesigned GSP that anneals to a site
located within the cDNA molecule the other is a universal amplification primer that targets the
mRNA of the cDNA complementary to the 3´ end of the mRNA Two universal amplification
primers are provided with the system The universal amplification primer (UAP) is designed for
the rapid and efficient cloning of RACE products using the uracil DNA glycosylase (UDG)
cloning method (13–16) The abridged universal amplification primer (AUAP) is homologous to
the adapter sequence used to prime first strand cDNA synthesis
Since the 3´ RACE System utilizes the poly(A) tail region as an initial priming site multiple
amplification products may be synthesized depending on the degree of specificity conferred
by the GSP To generate a specific amplification product the user may find it advantageous to
design a second nested GSP as recommended by Frohman et al (10) and reamplify the
RACE products this procedure is discussed in greater detail at the end of this chapter
Isolation of Total RNA
One of the most important factors preceding the synthesis of substantially fulllength cDNA is
the isolation of intact RNA The quality of the RNA dictates the maximum amount of sequence
information that can be converted into cDNA Thus it is important to optimize the isolation of
RNA from a given biological source and to prevent adventitious introduction of
RNases (17) and inhibitors of reverse transcriptase such as guanidinium salts SDS and
EDTA (18) RNA can be isolated using a variety of methods The recommended method for 3´
RACE is the guanidine isothiocyanateacidphenol method originally described by
Chomzynski and Sacchi (19) The TRIzol® Reagent method is an improvement of the original
singlestep method of Chomczynski and Sacchi (20) and can be used for the preparation of
RNA from as little as 103 cells or milligram quantities of tissue (21) Total RNA isolated with
TRIzol® Reagent is undegraded and essentially free of protein and DNA contamination For
the isolation of RNA from small quantities of sample (<106 cells or <10 mg tissue) without
using phenol the GlassMAX RNA Microisolation Spin Cartridge System is
recommended (22)
Total RNA isolated by these methods may contain small amounts of genomic DNA that may
subsequently be amplified along with the target cDNA The presence of this DNA is not likely
to cause problems because it lacks the poly(A) region present in the mRNA analyte As a
precaution however we recommend performing a control experiment without reverse
transcriptase to determine whether a given fragment is of genomic DNA or of cDNA origin
Products generated in the absence of RT are of genomic origin If your application requires
removal of all genomic DNA from your RNA preparation refer to DNase I Digestion of RNA
Preparation
First Strand cDNA Synthesis from Total RNA
The first strand cDNA synthesis reaction is catalyzed by SuperScript™ II RT This enzyme is
a mutant of MMLV RT that has been engineered to reduce RNase H activity resulting in
greater yields and more fulllength synthesis (232425) The enzyme exhibits increased
thermal stability and may be used at temperatures up to 50°C In addition SuperScript™ II
RT is not inhibited significantly by ribosomal and transfer RNA and may be used to synthesize
first strand cDNA from a total RNA preparation The RNA template is removed from the
cDNARNA hybrid molecule by digestion with RNase H after cDNA synthesis to increase the
sensitivity of PCR (26) The AP which primes first strand cDNA synthesis has been
engineered to contain three restriction endonuclease sites and a Not I halfsite Inclusion of
these sequences in the primer may facilitate postamplification cloning using either a
restriction endonucleasebased (27) or a T4 DNA polymerasebased (28) method Because
the AP initiates cDNA synthesis at the poly(A) region of the mRNA it effectively selects for
polyadenylated mRNAs thus oligo(dT)selection for poly(A)+ RNA is typically not necessary
although incorporating this step may facilitate the detection of rare mRNA transcripts
Amplification of a Target cDNA
TOP
Amplification of a target cDNA requires priming with two oligonucleotides and Taq DNA
polymerase The sense amplification primer is the userprovided GSPwhich is specific for the
particular gene or sequence of interest and may be designed to include sequence elements
that facilitate subsequent cloning steps The antisense amplification primer is one of the two
universal amplification primers provided with the system The AUAP contains a restriction
endonuclease site sequence (adapter region) homologous to the adapter region of the AP The
UAP is composed of the same adapter region plus a dUMPcontaining sequence at the 5´ end
of the primer required for UDGmediated cloning The UAP should not be used to prime DNA
synthesis with any archaeobacterial polymerase (eg Pyrococcus furiosus Pyrococcus woesei
etc) or any long PCR enzyme mixture (eg Elongase® Enzyme Mix) that contains one of these
enzymes due to the inhibition of polymerase activity by dUMPcontaining DNA Both the AUAP
and the UAP will function in PCR at annealing temperatures up to 68°C
Design of the GeneSpecific Primer
Efficient and specific PCR amplification is highly dependent on primer design This is especially
true for RACE applications since the PCR is carried out with only a single GSP In general
effective primers form stable duplexes with their target sequences are highly specific for their
target sequences and are free of secondary structure such as hairpin loops and dimers (29–
31) Additionally the complementarity of primer 3´termini must be minimized since primer
dimer artifacts may significantly reduce PCR efficiency Therefore dimer formation with the
AUAP or UAP primer as well as itself should be reduced Computer algorithms that have been
developed (32–35) and are commercially available often facilitate this analysis Discussion of
primer design for RACE applications may be found in Frohman (11) and Loh (6) It should be
noted that in cases where only limited peptide sequence information is available a degenerate
GSP may be prepared The AUAP and UAP included wit the system have been engineered
to function at PCR annealing temperatures up to 68°C and to facilitate the cloning step The
userdefined GSPs need to be compatible with the cloning method Add the following to the 5’
end of the GSP for UDG cloning 5´–CAU CAU CAU CAU–3´ (use with UAP) for T4 DNA
polymerase cloning 5´–CGA–3´ (use with AUAP)
Nested Amplification
The AP is designed to synthesize first strand cDNA from all polyadenylated mRNAs The
sequence specificity in the amplification reaction is therefore derived solely from the GSP Often
a second nested GSP may be utilized in conjunction with the AUAP or UAP in a second
amplification reaction to give the 3´ RACE procedure the specificity of a second primer(9) The
nested GSP can anneal immediately adjacent to the first GSP or at sequences within the cDNA
further downstream The nested amplification reaction may be conveniently conducted using a
plug of agarose from the gel analysis of the initial 3´ RACE reaction (see Nested Amplification
from an Agarose Plug) Ultimately the 3´ RACE procedure should produce a single prominent
band on an agarose gel When performing 3´ RACE with a nested primer sequences specific
for subsequent cloning manipulations (see Design of the GeneSpecific Primer) must be
designed into the nested GSP
Cloning of Amplification Products
Conventional cloning methods that typically involve endrepair and bluntend cloning can be
problematic for amplified products (36–38) An alternative is a rapid and efficient method
involving the use of UDG (13–16) This method requires that the user design a GSP containing
containing a 5'(CAU)4 sequence Incorporation of dUMP into the GSP may be accomplished
on most automated synthesizers or with Invitrogen Custom Primers (see Design of the Gene
Specific Primer) The product of the 3´ RACE reaction primed with the UAP and the dUMP
containing GSP is treated with UDG which converts dUMP residues to abasic sites (3940) to
generate 3´ overhangs The directional nature of the UDG cloning process can be exploited to
lend an added level of specificity to the RACE procedure Only amplification product that results
from priming by both the UAP and the appropriately designed GSP are efficient substrates for
UDG cloning Another alternative to conventional cloning methods uses the 3´ to 5´
exonuclease activity of T4 DNA polymerase as the basis for cloning as described by Stoker (28)
In this procedure the AUAP is used in the amplification reaction and the 3´ RACE products
are treated with T4 DNA polymerase to generate a Not I 5´ overhang Similarly the user may
design a site into the GSP (see Design of the GeneSpecific Primer) Another approach to
cloning is to digest the 3´ RACE product using one of the restriction endonuclease sites
designed into the AUAP The user may also design unique restriction sites into the GSP
exploit a site present in the cDNA sequence or endrepair the 3´ RACE product prior to
restriction endonuclease digestion(37)
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sequences from a messenger RNA template between a defined internal site and either the 3´
or the 5´ end of the mRNA (1) This methodology of amplification with singlesided specificity
has been described as onesided PCR (2) or anchored PCR (3) PCR requires two
sequencespecific primers that flank the sequence to be amplified (45) However to amplify
and characterize regions of unknown sequences this requirement imposes a limitation (3) 3´
RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for
PCR In this procedure mRNAs are converted into cDNA using reverse transcriptase (RT) and
an oligodT adapter primer Specific cDNA is then amplified by PCR using a genespecific
primer (GSP) that anneals to a region of known exon sequences and an adapter primer that
targets the poly(A) tail region This permits the capture of unknown 3´mRNA sequences that
lie between the exon and the poly(A) tail 5´ RACE uses an antisense gene specific primer for
the synthesis of specific cDNA by reverse transcriptase
Prior to PCR a TdTtailing step attaches an adapter sequence to the unknown 5´ sequences
of the cDNA Specific cDNA is then amplified by PCR using a GSP that anneals in a region of
known exon sequences and an adapter primer that targets the 5´ terminus RACE has been
used for amplification and cloning of rare mRNAs (6) and may be applied to existing cDNA
libraries (7) Additionally RACE products can be directly sequenced without any intermediate
cloning steps (89) or the products may be used to prepare probes (10) Products generated
by the 3´ and 5´ RACE procedures may be combined to generate fulllength cDNAs (1011)
Lastly the RACE procedures may be utilized in conjunction with exontrapping methods (12) to
enable amplification and subsequent characterization of unknown coding sequences
The 3´ RACE procedure is summarized as follows First strand cDNA synthesis is initiated at
the poly(A) tail of mRNA using the adapter primer (AP) After first strand cDNA synthesis the
original mRNA template is destroyed with RNase H which is specific for RNADNA
heteroduplex molecules Amplification is performed without intermediate organic extractions or
ethanol precipitations using two primers one is a userdesigned GSP that anneals to a site
located within the cDNA molecule the other is a universal amplification primer that targets the
mRNA of the cDNA complementary to the 3´ end of the mRNA Two universal amplification
primers are provided with the system The universal amplification primer (UAP) is designed for
the rapid and efficient cloning of RACE products using the uracil DNA glycosylase (UDG)
cloning method (13–16) The abridged universal amplification primer (AUAP) is homologous to
the adapter sequence used to prime first strand cDNA synthesis
Since the 3´ RACE System utilizes the poly(A) tail region as an initial priming site multiple
amplification products may be synthesized depending on the degree of specificity conferred
by the GSP To generate a specific amplification product the user may find it advantageous to
design a second nested GSP as recommended by Frohman et al (10) and reamplify the
RACE products this procedure is discussed in greater detail at the end of this chapter
Isolation of Total RNA
One of the most important factors preceding the synthesis of substantially fulllength cDNA is
the isolation of intact RNA The quality of the RNA dictates the maximum amount of sequence
information that can be converted into cDNA Thus it is important to optimize the isolation of
RNA from a given biological source and to prevent adventitious introduction of
RNases (17) and inhibitors of reverse transcriptase such as guanidinium salts SDS and
EDTA (18) RNA can be isolated using a variety of methods The recommended method for 3´
RACE is the guanidine isothiocyanateacidphenol method originally described by
Chomzynski and Sacchi (19) The TRIzol® Reagent method is an improvement of the original
singlestep method of Chomczynski and Sacchi (20) and can be used for the preparation of
RNA from as little as 103 cells or milligram quantities of tissue (21) Total RNA isolated with
TRIzol® Reagent is undegraded and essentially free of protein and DNA contamination For
the isolation of RNA from small quantities of sample (<106 cells or <10 mg tissue) without
using phenol the GlassMAX RNA Microisolation Spin Cartridge System is
recommended (22)
Total RNA isolated by these methods may contain small amounts of genomic DNA that may
subsequently be amplified along with the target cDNA The presence of this DNA is not likely
to cause problems because it lacks the poly(A) region present in the mRNA analyte As a
precaution however we recommend performing a control experiment without reverse
transcriptase to determine whether a given fragment is of genomic DNA or of cDNA origin
Products generated in the absence of RT are of genomic origin If your application requires
removal of all genomic DNA from your RNA preparation refer to DNase I Digestion of RNA
Preparation
First Strand cDNA Synthesis from Total RNA
The first strand cDNA synthesis reaction is catalyzed by SuperScript™ II RT This enzyme is
a mutant of MMLV RT that has been engineered to reduce RNase H activity resulting in
greater yields and more fulllength synthesis (232425) The enzyme exhibits increased
thermal stability and may be used at temperatures up to 50°C In addition SuperScript™ II
RT is not inhibited significantly by ribosomal and transfer RNA and may be used to synthesize
first strand cDNA from a total RNA preparation The RNA template is removed from the
cDNARNA hybrid molecule by digestion with RNase H after cDNA synthesis to increase the
sensitivity of PCR (26) The AP which primes first strand cDNA synthesis has been
engineered to contain three restriction endonuclease sites and a Not I halfsite Inclusion of
these sequences in the primer may facilitate postamplification cloning using either a
restriction endonucleasebased (27) or a T4 DNA polymerasebased (28) method Because
the AP initiates cDNA synthesis at the poly(A) region of the mRNA it effectively selects for
polyadenylated mRNAs thus oligo(dT)selection for poly(A)+ RNA is typically not necessary
although incorporating this step may facilitate the detection of rare mRNA transcripts
Amplification of a Target cDNA
TOP
Amplification of a target cDNA requires priming with two oligonucleotides and Taq DNA
polymerase The sense amplification primer is the userprovided GSPwhich is specific for the
particular gene or sequence of interest and may be designed to include sequence elements
that facilitate subsequent cloning steps The antisense amplification primer is one of the two
universal amplification primers provided with the system The AUAP contains a restriction
endonuclease site sequence (adapter region) homologous to the adapter region of the AP The
UAP is composed of the same adapter region plus a dUMPcontaining sequence at the 5´ end
of the primer required for UDGmediated cloning The UAP should not be used to prime DNA
synthesis with any archaeobacterial polymerase (eg Pyrococcus furiosus Pyrococcus woesei
etc) or any long PCR enzyme mixture (eg Elongase® Enzyme Mix) that contains one of these
enzymes due to the inhibition of polymerase activity by dUMPcontaining DNA Both the AUAP
and the UAP will function in PCR at annealing temperatures up to 68°C
Design of the GeneSpecific Primer
Efficient and specific PCR amplification is highly dependent on primer design This is especially
true for RACE applications since the PCR is carried out with only a single GSP In general
effective primers form stable duplexes with their target sequences are highly specific for their
target sequences and are free of secondary structure such as hairpin loops and dimers (29–
31) Additionally the complementarity of primer 3´termini must be minimized since primer
dimer artifacts may significantly reduce PCR efficiency Therefore dimer formation with the
AUAP or UAP primer as well as itself should be reduced Computer algorithms that have been
developed (32–35) and are commercially available often facilitate this analysis Discussion of
primer design for RACE applications may be found in Frohman (11) and Loh (6) It should be
noted that in cases where only limited peptide sequence information is available a degenerate
GSP may be prepared The AUAP and UAP included wit the system have been engineered
to function at PCR annealing temperatures up to 68°C and to facilitate the cloning step The
userdefined GSPs need to be compatible with the cloning method Add the following to the 5’
end of the GSP for UDG cloning 5´–CAU CAU CAU CAU–3´ (use with UAP) for T4 DNA
polymerase cloning 5´–CGA–3´ (use with AUAP)
Nested Amplification
The AP is designed to synthesize first strand cDNA from all polyadenylated mRNAs The
sequence specificity in the amplification reaction is therefore derived solely from the GSP Often
a second nested GSP may be utilized in conjunction with the AUAP or UAP in a second
amplification reaction to give the 3´ RACE procedure the specificity of a second primer(9) The
nested GSP can anneal immediately adjacent to the first GSP or at sequences within the cDNA
further downstream The nested amplification reaction may be conveniently conducted using a
plug of agarose from the gel analysis of the initial 3´ RACE reaction (see Nested Amplification
from an Agarose Plug) Ultimately the 3´ RACE procedure should produce a single prominent
band on an agarose gel When performing 3´ RACE with a nested primer sequences specific
for subsequent cloning manipulations (see Design of the GeneSpecific Primer) must be
designed into the nested GSP
Cloning of Amplification Products
Conventional cloning methods that typically involve endrepair and bluntend cloning can be
problematic for amplified products (36–38) An alternative is a rapid and efficient method
involving the use of UDG (13–16) This method requires that the user design a GSP containing
containing a 5'(CAU)4 sequence Incorporation of dUMP into the GSP may be accomplished
on most automated synthesizers or with Invitrogen Custom Primers (see Design of the Gene
Specific Primer) The product of the 3´ RACE reaction primed with the UAP and the dUMP
containing GSP is treated with UDG which converts dUMP residues to abasic sites (3940) to
generate 3´ overhangs The directional nature of the UDG cloning process can be exploited to
lend an added level of specificity to the RACE procedure Only amplification product that results
from priming by both the UAP and the appropriately designed GSP are efficient substrates for
UDG cloning Another alternative to conventional cloning methods uses the 3´ to 5´
exonuclease activity of T4 DNA polymerase as the basis for cloning as described by Stoker (28)
In this procedure the AUAP is used in the amplification reaction and the 3´ RACE products
are treated with T4 DNA polymerase to generate a Not I 5´ overhang Similarly the user may
design a site into the GSP (see Design of the GeneSpecific Primer) Another approach to
cloning is to digest the 3´ RACE product using one of the restriction endonuclease sites
designed into the AUAP The user may also design unique restriction sites into the GSP
exploit a site present in the cDNA sequence or endrepair the 3´ RACE product prior to
restriction endonuclease digestion(37)
《香当网》用户分享的内容，不代表《香当网》观点或立场，请自行判断内容的真实性和可靠性！
该内容是文档的文本内容，更好的格式请下载文档