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Overview

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 unknown sequences at either the 3' or the 5' -end of the mRNA (1). This methodology of amplification with single-sided specificity has been described by others as “one-sided” PCR (2) or “anchored” PCR (3).

In general, PCR amplification of relatively few target molecules in a complex mixture requires two sequence-specific primers that flank the region of sequence to be amplified (4,5). However, to amplify and characterize regions of unknown sequences, this requirement imposes a severe limitation(3). 3' and 5' RACE methodologies offer possible solutions to this problem. 3' RACE takes advantage of the natural poly(A) tail in mRNA as a generic priming site for PCR amplification.

In this procedure, mRNAs are converted into cDNA using reverse transcriptase (RT) and an oligo-dT adapter primer. Specific cDNA is then directly amplified by PCR using a gene-specific 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, or “anchored” PCR, is a technique that facilitates the isolation and characterization of 5' ends from low-copy messages. The method has been reviewed by both Frohman (6,8) and Loh (7).

Although the precise protocol varies among different users, the general strategy remains consistent. First strand cDNA synthesis is primed using a gene-specific antisense oligonucleotide (GSP1). This permits cDNA conversion of specific mRNA, or related families of mRNAs, and maximizes the potential for complete extension to the 5' -end of the message. Following cDNA synthesis, the first strand product is purified from unincorporated dNTPs and GSP1. TdT (Terminal deoxynucleotidyl transferase) is used to add homopolymeric tails to the 3' ends of the cDNA. In the original protocol, tailed cDNA is then amplified by PCR using a mixture of three primers: a nested gene-specific primer (GSP2), which anneals 3' to GSP1; and a combination of a complementary homopolymer-containing anchor primer and corresponding adapter primer which permit amplification from the homopolymeric tail. This allows amplification of unknown sequences between the GSP2 and the 5'-end of the mRNA. RACE procedures have been used for amplification and cloning of rare mRNAs that may escape, or prove challenging for, conventional cDNA cloning methodologies (7). Additionally, RACE may be applied to existing cDNA libraries (9). Random hexamerprimed cDNA has also been adapted to 5' RACE for amplification and cloning of multiple genes from a single first strand synthesis reaction (10). Products of RACE reactions can be directly sequenced without any intermittent cloning steps (11,12), or the products can be used for the preparation of probes (13). Products generated by the 3' and 5' RACE procedures may be combined to generate full-length cDNAs (6,13). Lastly, the RACE procedures may be utilized in conjunction with exon trapping methods (14) to enable amplification and subsequent characterization of unknown coding sequences.

Summary of the 5' RACE System

The 5' RACE System is a set of prequalified reagents intended for synthesis of first strand cDNA, purification of first strand products, homopolymeric tailing, and preparation of target cDNA for subsequent amplification by PCR. Control RNA, DNA, and primers are provided for monitoring system performance.


First strand cDNA is synthesized from total or poly(A)+ RNA using a gene-specific primer (GSP1) that the user provides and Invitrogen™ SuperScript™ II Reverse Transcriptase (RT), a derivative of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) with reduced RNase H activity. After first strand cDNA synthesis, the original mRNA template is removed by treatment with the RNase Mix (mixture of RNase H, which is specific for RNA:DNA heteroduplex molecules, and RNase T1). Unincorporated dNTPs, GSP1, and proteins are separated from cDNA using a S.N.A.P. Column. A homopolymeric tail is then added to the 3'-end of the cDNA using TdT and dCTP.

Since the tailing reaction is performed in a PCR-compatible buffer, the entire contents of the reaction may be directly amplified by PCR without intermediate organic extractions, ethanol precipitations, or dilutions. PCR amplification is accomplished using Taq DNA polymerase, a nested, gene-specific primer (GSP2, designed by the user) that anneals to a site located within the cDNA molecule, and a novel deoxyinosine-containing anchor primer (patent pending) provided with the system.

Following amplification, 5' RACE products can be cloned into an appropriate vector for subsequent characterization procedures, which may include sequencing, restriction mapping, preparation of probes to detect the genomic elements associated with the cDNA of interest, or in vitro RNA synthesis. The Abridged Anchor Primer (AAP), Abridged Universal Amplification Primer (AUAP), Anchor Primer (AP) [available separately], and Universal Amplification Primer (UAP) include recognition sequences for Mlu I, Sal I, and Spe I to facilitate restriction endonuclease cloning of RACE products.

5' RACE products may also be used for procedures that do not require an intermittent cloning step such as dsDNA cycle sequencing (12,18) or probe preparation (13). However, additional rounds of PCR using the AUAP, or UAP, in conjunction with either progressively nested GSPs or size-selected products from the initial PCR, may be required to confer an adequate level of specificity to the process to permit direct characterization of RACE products. Details of the individual steps of 5' RACE are discussed below. Review this information carefully before beginning. The RNA isolation, design of primers, and the amplification protocols are most important for optimal results.

Isolation of 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 (19), and to prevent introduction of RNases and inhibitors of RT (20, 52). The guanidine isothiocyanate/acid-phenol method, originally described by Chomzynski and Sacchi (21), is the recommended method for RNA isolation.

Design of 5' RACE primers

The sensitivity and the specificity of the first strand synthesis and the subsequent PCR depend upon good primer design. A minimum of two antisense gene-specific primers (GSP) are required for 5' RACE and must be supplied by the user.

  1. GSP1 primes first-strand cDNA synthesis. Design this primer to anneal at least 300 bp from the mRNA 5'-end so that the cDNA can be easily purified using a S.N.A.P. column.
  2. A second nested primer, GSP2, that anneals to sequences located 3' (with respect to cDNA not mRNA) of GSP1 is required for PCR. GSP2 can anneal immediately adjacent to GSP1 or at sequences located further upstream of GSP1 within the cDNA product. Other sequentially nested GSPs may be required depending on the efficiency and specificity of the primary PCR.


In general, these primers should be highly specific for their target sequences, able to form stable duplexes with their target sequences, and free of secondary structure. The key rules for primer design are discussed below (as well as in references 22- 26). The primers provided in this system were carefully designed for successful 5' RACE. The anchor primers contain 3' sequence complementary to the homopolymeric tail and additional 5' sequence that encodes an adapter region, comprised of restriction endonuclease sites and other functional sequences which facilitate cloning and characterization of 5' RACE products. Normally, homopolymer primers create melting temperatures that are either higher [poly (dG)•poly (dC)] or lower [poly (dA)•poly (dT)] than a typical GSP. They also can have poor specificity that can lead to mispriming at internal sequences. To minimize these problems, our anchor primers were designed with the selective placement of deoxyinosine residues in the poly (dG) portion. This design eliminates the need to use the mixtures of anchor and adapter primers described in the original method (6,7).

  • Note:   While the anchor primers enable efficient amplification of many target sequences, they may not be an idealized solution for all 5' RACE applications. It is possible that the anchor primer may anneal at certain gene-specific sequences. Therefore, as in any RACE procedure, specificity of the anchor primer for the oligo-dC tail should be tested by performing amplification reactions with cDNA subjected to dC-tailing both in the presence of and absence of TdT.


Deoxyinosine has the capacity to base-pair with all four bases; however, it does so with varying affinities. The order of stabilities for the different combinations, from greatest to least stable, reported by Martin et al. are as follows: I:C, I:A, I:T, and I:G. I:C pairs were found to be slightly less stable than A:T pairs (27). The selective placement of deoxyinosine residues in the 3' region of the anchor primer maintains low stability on the primer’s 3'-end (ΔG = -8.2 kCal/mol) and creates a melting temperature (Tm) for the 16-base anchor region (66.6°C) which is comparable to that of a typical 20-mer primer with 50% GC content (22,23). This maximizes specific priming from the oligo-dC tail, minimizes priming at internal C-rich regions of the cDNA, and establishes a relationship of a “balanced” Tm for the anchor region to that of GSP2, which is required for efficient PCR (6,7).

The Abridged Universal Amplification Primer (AUAP) and Universa  Amplification Primer (UAP) are used to reamplify primary 5' RACE PCR products in applications such as nested PCR or enrichment of RACE products for cloning. The AUAP contains a restriction endonuclease site sequence (adapter region) homologous to the adapter region of the anchor primer. The UAP is composed of the same adapter region as the AUAP plus a dUMP-containing sequence at the 5'-end of the primer required for uracil DNA glycosylase (UDG)-mediated cloning of 5' RACE products. The original 5' RACE Anchor Primer is available separately for applications that require UDG cloning of 5' RACE products directly from the primary PCR. The UAP, the 5' RACE Anchor Primer or any dUMP-containing primer should not be used to prime DNA synthesis with any archaeobacterial polymerase (Pfu DNA Polymerase, Pwo DNA Polymerase, etc.), including long PCR enzyme mixtures (28,29), because dUMP inhibits these polymerase activities.

First strand cDNA synthesis from total RNA

The capture of mRNA 5'-ends is dependent on complete cDNA synthesis. The use of RNase H- RT for first strand synthesis results in greater full-length cDNA synthesis and higher yields of first strand cDNA than obtained with other RTs (20,30). SuperScript II RT has been engineered to retain the full DNA polymerase activity found in M-MLV RT (31). The enzyme exhibits increased thermal stability and may be used at temperatures up to 50°C. Because SuperScript™ II RT is not inhibited significantly by ribosomal and transfer RNA, it may be used effectively to synthesize first strand cDNA from a total RNA preparation. The RNA template is removed from the first strand cDNA product as described below.

Removal of RNA template by RNase Mix

After cDNA synthesis, RNase Mix, a mixture of RNase H and RNase T1, is used to degrade the RNA. The digestion is performed following thermal inactivation of the RT in order to reduce the potential for hairpin-primed second-strand synthesis (catalyzed by RT)  which can obscure the accessibility of the cDNA ends to TdT. Template RNA in the cDNA:RNA hybrid is degraded by RNase H and the single-stranded RNAs are degraded by RNase T1. This eliminates possible renaturation of template RNA to cDNA. TdT does not use RNA as a substrate (32); however, RNA may inhibit tailing and subsequent PCR of the cDNA (33). Use of the RNase Mix upon completion of first strand synthesis is crucial to the efficiency of the tailing reaction because TdT exhibits a marked preference for single-stranded substrates (34,35).


Purification of first strand product

Excess nucleotides and GSP1 must be removed from the first strand product. Otherwise, residual GSP1 will be tailed by TdT and will compete for Abridged Anchor Primer during PCR. Because of the large amounts of GSP1 relative to cDNA product, a stringent purification procedure is required (6,7,36). The S.N.A.P. column procedure, adapted from a method described by Vogelstein and Gillespie (37), provides a rapid and efficient means to purify first strand product. In the presence of the chaotropic agent, sodium iodide, cDNA >200 bases ar  bound to the silica-based membrane. Buffer components, dNTPs, enzymes, and oligonucleotides remain in solution and are removed by centrifugation with the effluent. Residual impurities and sodium iodide are removed by passing several volumes of 1X wash buffer followed by a 70% ethanol rinse through the S.N.A.P. column. Purified cDNA is recovered in distilled water and may be used directly in the TdT tailing reaction.

Homopolymeric tailing of cDNA

TdT tailing creates the abridged anchor primer binding site on the 3'-end of the cDNA. Efficient tailing is necessary to provide:

  1.    A high proportion of tailed cDNA molecules for efficient amplification of first strand products.

  2.    Homopolymeric tails of sufficient length to allow the primer to anneal.

  3.    Homopolymeric tails of uniform length to produce a homogeneous amplification product.

  4.    A buffer compatible with the PCR buffer system.

The 5' RACE System tailing reaction has been optimized to meet these criteria. The 5' RACE System uses a tailing buffer [10 mM Tris-HCl (pH 8.4), 25 mM KCl and 1.5 mM MgCl2] supplemented with 200 μM dCTP for homopolymeric tailing of first strand cDNA. The tailing reaction is highly sensitive to the concentration of each buffer component.

Concentrations of MgCl2; in excess of 1.5 mM may significantly inhibit both the length of the tail and the percentage of molecules tailed. In general, components such as Tris buffers and salts have been reported to be inhibitory to TdT (32), and CoCl2 has classically been chosen over MgCl2 as the optimal divalent cation for tailing reactions (35). However, careful manipulation of buffers containing these components has been shown to produce results that are highly effective for 5' RACE (36).

Double-stranded 3' termini and hairpin structures may significantly impair homopolymeric tailing of cDNA; therefore, a brief denaturation procedure prior to tailing is used to disrupt any secondary structure in the cDNA.

The choice of nucleotide for homopolymeric tailing has been a subject of debate. Each nucleotide offers unique advantages and disadvantages. The 5' RACE System uses dCtailing to complement our unique Abridged Anchor Primer. dA-tailing permits the use of the same oligo-dT anchor primer for both 5' and 3' RACE procedures. However, because A:T base pairs are less stable than G:C base pairs, longer stretches of dAs or dT  are required for priming as compared to dGs or dCs.

Amplification of target cDNA

Successful 5' RACE is extremely dependent on the efficiency and specificity of the PCR. Optimal conditions for amplification are dependent on the nature of each particular primer and target sequence used. Alteration of the magnesium ion, dNTP, or primer concentration, as well as the thermal cycling protocol, may be required. The optimal free magnesium concentration for efficient amplification is reported to be between 0.7 and 0.8 mM (38). Since magnesium can bind deoxynucleoside triphosphates, this factor is affected by both primer and dNTP concentration. In general, lower concentrations of dNTP (50 to 200 μM), MgCl2 (1 to 1.5 mM), and primer (0.1 to 0.2 μM) promote higher fidelity and product yield as well as to promote 3'-terminal T-mismatches (40). This factor may warrant consideration if a degenerate oligonucleotide is used as GSP2. Additionally, since a degenerate primer represents a composite of many different priming sequences, higher primer concentrations (≥ μM) are generally required. Typical thermal cycling parameters are provided in the protocols. However, optimal conditions depend not only on the primers and template, but also the type of PCR tube as well as the thermal cycler.

There are four times and temperatures that must be considered:

  1)    Preamplification denaturation of the template DNA (PAD)

  2)    Denaturation of product DNA at the beginning of each cycle

  3)    Annealing of the primers to the denatured DNA

  4)    Extension of the primers by the polymerase

Steps 2-4 constitute a cycle and are repeated usually 30-35 times followed by a final extension time of 5-10 min and then a holding temperature of 5°C.

Many PCR protocols use a PAD step of 3 to 5 min. However, an extended PAD is not usually necessary and may impair the ability to amplify longer sequences (41). The denaturation temperature and time should be sufficient to completely separate target strands, yet minimized to reduce deamination and depurination of target DNA. For thin-walled tubes in thermal cyclers which use the sample temperature (or calculated sample temperature) to control temperature cycling, a denaturation time of 10 s to 15 s at 94°C is adequate. Likewise, an annealing time of 20 s to 30 s is usually ample. In contrast, PCR in conventional 0.5-ml microcentrifuge tubes may require 1 min for complete denaturation and 30 s to 1 min for annealing.

Optimal annealing temperature is dependent upon the thermodynamic properties of the primers. However, well-designed primers, i.e. primers with unstable 3'-ends (ΔG > -9 kCal/mol), can function effectively in PCR over a broad range of annealing temperatures (41). A general rule for extension time is to allow 1 min for every 1 kb of target sequence. If primer Tms are ≥68°C, a two step PCR, which cycles between denaturation at 94°C and combined annealing and extension at 68°C can be used. For a detailed discussion of parameters affecting PCR, please refer to Innis and Gelfand (42) or Saiki (38, 43).

Nonspecific annealing and extension of primers prior to the initial denaturation step of the PCR process may adversely affect the efficiency and specificity of amplification. These artifacts can be minimized by using the “hot start” technique (44,45) which requires the addition of either Taq DNA polymerase, dNTPs, or MgCl2 after reactions have been equilibrated at 75°C to 80°C. For many applications it is sufficient to assemble the reactions on ice, in thin-walled PCR tubes and directly transfer the tubes to a thermal cycler equilibrated to the initial denaturation temperature, 94°C.

Amplification of a target cDNA synthesized with the 5' RACE System requires priming with two oligonucleotides. The Abridged Anchor Primer, which is specific for the oligodC tail added by Td  serves as the sense primer. The antisense primer (GSP2), provided by the user, should anneal to an internal (nested) site within the cDNA sequence (with respect to the primer used for first strand synthesis, GSP1) and may include sequence elements that facilitate subsequent cloning steps.

Use of a nested GSP2 is essential for effective PCR (6,7,36). This not only adds a level of specificity to the process, but it prevents “primer-dimer” amplification of residual GSP1 that may carry through the cDNA purification procedure.  Residual GSP1, which is subsequently tailed by TdT, is copied by extension of the anchor primer during PCR. This results in amplification of the tailed GSP1 sequence and blocks amplification of cDNA.

Cloning 5' RACE amplification products

Conventional cloning methods that typically involve end-repair and blunt-end cloning can be problematic for amplified products (18,46,47). An alternative is a rapid and efficient cloning method involving the use of UDG (48-50). This method requires that the user design a nested GSP2 containing a 5'-(CAU)4 sequence. Incorporation of dUMP into the nested GSP2 may be accomplished with minimal expense on most automated synthesizers or by ordering through Invitrogen’s Custom Primers. An 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 (51). In this procedure, PCR products from the primary PCR with the Abridged Anchor Primer, or nested amplification reaction primed with the AUAP, are treated with T4 DNA polymerase to generate a Not I 5' overhang. Another approach to cloning is to digest the 5' RACE product using one of the restriction endonuclease sites designed into the AUAP  (1). The user may also design unique restriction sites into the GSP, exploit a site present in the cDNA sequence or end-repair the 5' RACE product prior to restriction-endonuclease digestion (46).

Methods

Components and storage 

The components of the 5' RACE System are as follows. Sufficient material is provided for 10 reactions. One reaction prepares specific cDNA from 1-5 μg of a total RNA or 50- 500 ng poly (A)+ RNA preparation for amplification by anchored PCR. The amount of RNA will vary depending on the application. Control RNA, DNA, and primers are included to verify the performance of the system and may be added to experimental RNA preparations to monitor the efficiency of each step or to troubleshoot potential problems.

ComponentVolumeStorage
Reagents:  
10X PCR buffer
[200 mM Tris-HCl (pH 8.4), 500 mM KCl]		500 μl–20°C
25 mM MgCl2500 μl –20°C   
10 mM dNTP mix
10 mM each dATP, dCTP, dGTP, dTTP]		100 μl–20°C
0.1 M DTT100 μl–20°C
SuperScript II Reverse Transcriptase
(200 units/μl)		10 μl–20°C
RNase mix10 μl–20°C
5X tailing buffer [50 mM Tris-HCl (pH 8.4),
125 mM KCl, 7.5 mM MgCl2]		500 μl–20°C
2 mM dCTP50 μl–20°C
 terminal deoxynucleotidyl transferase15 μl–20°C
5' RACE abridged anchor primer (AAP, 10 μM)80 μl–20°C
universal amplification primer (UAP, 10 μM)40 μl–20°C
abridged universal amplification primer (AUAP, 10 μM)40 μl–20°C
DEPC-treated water1.25 ml   –20°C
control gene-specific primer 1 (GSP1, 1 μM)25 μl–20°C
control nested gene-specific primer 2
(GSP2, 10 μM)		80 μl–20°C
control PCR primer, gene-specific primer 3
(GSP3, 10 μM)		20 μl–20°C
control DNA (2 x 104 copies/μl; ~0.1 pg/μl)100 μl–20°C
control RNA (50 ng/μl)10 μl–70°C
DNA purification system:  
S.N.A.P. Columns10 columns4°C
Collection tubes10 tubes4°C
binding solution (6 M sodium iodide)30 ml4°C
Wash buffer concentrate30 ml4°C
  • Note:  The 10X PCR buffer does not contain MgCl2. Therefore, MgCl2 must be added to the first strand reaction mix.
  • Note:  Do not freeze the DNA Purification System


Additional materials required

The following items are required for use with the 5' RACE System, but are not included.

  • sterilized, RNase-free thin-walled 0.2 or 0.5-ml PCR tubes;
  • automatic pipettes capable of dispensing 1 to 20 μl and 20 to 200 μl;
  • sterilized, RNase-free disposable tips for automatic pipettes;
  • disposable latex gloves;
  • sterilized, distilled water;
  • absolute ethanol;
  • GSP1 (cDNA primer, user-defined);
  • GSP2 (nested primer for PCR amplification of dC-tailed cDNA, user-defined, appropriately engineered);
  • microcentrifuge capable of generating a relative centrifugal force of 13,000 x g;
  • 37°C, 42°C, 65°C, and 70°C water baths or heat blocks (or use thermal cycler);
  • Taq DNA Polymerase;
  • programmable thermal cycler; and
  • mineral oil (if necessary for your thermal cycler)


Performance and limitations of procedures

The 5' RACE System has been functionally tested using the control RNA according to the protocols described in this manual using GIBCO BRL Taq DNA Polymerase. Following PCR, a distinct 711-bp band was visible by agarose gel electrophoresis and ethidium bromide staining. While the 5' RACE system provides a direct and reliable solution for the preparation of tailed cDNA, PCR with single-sided specificity remains highly challenging. Success with the system is extremely dependent on the efficiency and specificity of the PCR used to amplify your tailed cDNA. Taq DNA polymerase from other suppliers may not function as well in the buffers provided in this system. The PCR protocol is intended as a starting point. Optimal amplification parameters for your target gene may vary.

Obtaining longer 5' RACE products, i.e. greater than 1 kb, adds an additional challenge to the procedure. The 5' RACE System has been used successfully with Invitrogen™ eLONGase™ Enzyme Mix, an enzyme system designed for amplification of long templates, to obtain an increased yield of amplification product as well as substantially increased length of 5' RACE products. The principle barrier to long(er) 5' RACE lies in the specificity and efficiency of PCR. Critical success factors include primer design, PCR optimization, and a systematic experimental strategy that includes amplification of primary PCR using nested, gene-specific primers. Truncated products can yield informative sequence data that can be applied in additional 5' RACE experiments as one walks toward the 5'-end.

Advance preparations
Please review the advance preparation guidelines discussed in this section prior to starting to work with the 5' RACE System. To achieve optimal results, it is also recommended that you review above before using this system.

 

Isolation of total RNA

One of the most important factors affecting the synthesis of substantially full-length cDNA is the isolation of intact RNA. Therefore, it is important to optimize the isolation of RNA and to prevent introduction of RNases (19) and inhibitors of RT such as guanidinium salts, SDS and EDTA (20,52). The recommended method of RNA isolation is the guanidine isothiocyanate/acid-phenol method originally described by Chomzynski and Sacchi (21).The Invitrogen™ TRIzol™ Reagent method (53) is an improvement of the original single-step method of Chomczynski and Sacchi and can be used for the preparation of RNA from as little as 10³ cells (54). Total RNA isolated with TRIzol Reagent is undegraded and essentially free of protein and DNA contamination. To maintain intact RNA, an RNase-free environment is critical.

  • Note:   All temperature incubations may be performed in an appropriately programmed thermal cycler. This eliminates the need for multiple fixed temperature baths or heat blocks.


Total RNA may contain small amounts of genomic DNA that may be amplified along with the target cDNA. The presence of this double-stranded DNA is not likely to cause problems in 5' RACE, since it is inefficiently tailed prior to amplification. As a precaution, however, perform a control experiment without RT. 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 corresponding section. Oligo(dT)-selection for poly(A)+ RNA is typically not necessary although incorporating this step may facilitate the detection of rare mRNA transcripts.

Design of the gene-specific primers

Efficient and specific PCR is highly dependent on effective primer design. This is especially true for RACE applications since the PCR is carried out with only a single GSP. No method of primer design can guarantee successful amplification, so all primers must be tested in PCR before they can be pronounced “good”.

Primers for PCR (GSP2 AND GSP3): Effective primers form stable, highly specific duplexes with their target sequences, and are free of secondary structure such as hairpin loops and dimers (22,23,25,55). High stability, i.e., G:C clamps, in the 5'- and central regions of the primer confer hybridization stability with the target sequence. Primers with unstable 3'-ends (ΔG > -9 kCal/mol) often result in higher specificity, since the potential to misprime at nontarget sites is reduced. Generally, this condition can be met by including no more than two G or C residues in the last five 3'-bases (22,23). Additionally, 3'-terminal complementarity should be minimized since primerdimer artifacts may significantly reduce PCR efficiency. Therefore the nested amplification primer should be examined for dimer formation with the anchor primer, as well as itself.

Computer algorithms that have been developed (22,56-58) often facilitate this analysis as well as secondary structure analysis.

The next important parameter for primers is the Tm (the temperature at which 50% of the primer and its complementary sequence are present in a duplex DNA molecule.) The Tm is necessary to establish an annealing temperature for PCR. The annealing temperature should be low enough to guarantee efficient annealing of the primer to the target, but high enough to minimize nonspecific binding. Since a single GSP is used in RACE, use as stringent an annealing temperature as possible to minimize amplification of nonspecific products. As a good starting point, choose an annealing temperature a few degrees below the estimated Tm of the primer pair. In practice, primers with Tms between 60°C and 75°C usually can function effectively in 5' RACE. Ideally, the Tm of primers used for PCR should be closely matched (6). Several methods are available to estimate the Tm of primers. These provide only estimates and the optimal annealing temperature must be established experimentally. When designing UDG cloning primers, consider dU residues as if they were dT residues for the calculations.

Primer for first strand cDNA synthesis (GSP1):

For GSP1, follow the same rules used for PCR primer selection. The Tm should be appropriate for the relatively low temperature (42°C) of the cDNA synthesis reaction. A short primer (16 to 20 bases) facilitates efficient separation of GSP1 from cDNA product. Efficient recovery of cDNA from the S.N.A.P. column requires a product of at least 200 bases in length. Therefore, we recommend that GSP1 anneal to sequences located at least 300 bases from the mRNA 5'-end. Again, even well-designed primers may not function efficiently in priming first strand synthesis. For example, secondary structure of the mRNA at the annealing site may prevent efficient annealling or extension of the primer.

Primers for subsequent cloning:

The user defined GSPs need to be compatible with the cloning method. Add the following to the 5'-end of the nested GSP: for T4 DNA polymerase cloning 5'–CGA–3' (use with AUAP) for restriction endonuclease cloning, an appropriate adapter region (59) is required. It should be noted that in cases where only limited peptide sequence information is available, degenerate GSPs may be prepared. An alternative to the synthesis of degenerate primers is the substitution of dI residues at wobble-base positions (60-62). 13

1X wash buffer for S.N.A.P. procedure

Prior to using the system for the first time, a 1X wash buffer must be prepared from the wash buffer concentrate.

  1.    Pipette 1 ml of the wash buffer concentrate into a 50-ml graduated cylinder.

  2.    Add 18 ml of distilled water and 21 ml of absolute ethanol. Mix thoroughly.

  3.    Transfer to an appropriate-sized glass bottle. Cap and store at 4°C.

70% ethanol wash for S.N.A.P. procedure

  1.    Add 35 ml of absolute ethanol and 15 ml of distilled water to a 50-ml graduated cylinder.

  2.    Transfer to an appropriate-sized glass bottle. Cap and store at 4°C.

First strand cDNA synthesis

You may wish to use this protocol to familiarize yourself with the procedure before attempting 5' RACE with your sample. This procedure is designed to convert specific RNA sequence(s) from a background of 1-5 μg of total RNA into first strand cDNA. In general, 100 to 500 ng of total RNA should provide sufficient material for the amplification of low copy messages by 5' RACE (6). Although poly(A)+ RNA may be used in this protocol to enrich for very rare messages, this level of purity is typically not necessary.

  1. Add the following to a 0.5-ml microcentrifuge tube (or thin-walled PCR tube if using a thermal cycler):
    ComponentAmount
    GSP12.5 pmoles (~10 to 25 ng)
    Sample RNA
    		1-5 μg
    DEPC-treated water
    (or sterile, distilled water)    		sufficient for a final volume of 15.5 μl


    • Note:   Extreme care should be taken to avoid contamination of samples by RNase.
    • Note:   If you choose to use placental RNase inhibitor in the reaction, it should be added after the addition of DTT. Appropriate volume adjustments should be made in step 1. See precautionary notes regarding the use of RNase inhibitor proteins
    • Note:   For high GC content mRNA, use the Alternate Protocol to help reduce interference by secondary structure of mRNA.

     
  2. Incubate the mixture 10 min at 70°C to denature RNA. Chill 1 min on ice. Collect the contents of the tube by brief centrifugation and add the following in the order given:

    ComponentVolume (μl)
    10X PCR buffer2.5
    25 mM MgCl22.5
    10 mM dNTP mix1
    0.1 M DTT2.5
    Final Volume8.5

    The final volume of step 1 and 2 is 24 μl.
  3. Mix gently, and collect the reaction by brief centrifugation. Incubate for 1 min at 42°C.
  4. Add 1 μl of SuperScript™ II RT. Mix gently and incubate for 50 min at 42°C.

    Note:   30 min incubation is usually sufficient for short (<4 kb) mRNAs. Longer transcripts require at least 50 min to synthesize enough cDNA for a consistent signal in long PCR.

    Final composition of the reaction:

    20 mM Tris-HCl (pH 8.4)
    50 mM KCl
    2.5 mM MgCl2
    10 mM DTT
    100 nM cDNA primer (GSP1)
    400 μM each dATP, dCTP, dGTP, dTTP
    1-5 μg (~40 ng/μl) RNA
    200 units SuperScript II RT


    Note:   Keep enzymes on ice during the procedure. Mix, and quickly centrifuge each component before use.

    Note:   If you have >5 μg of total RNA, increase reaction volumes and amount of SuperScript II RT proportionately. If you have <1 μg of total RNA, no changes to the protocol are necessary. 50 to 500 ng of poly(A)+ RNA may be substituted for total RNA in this protocol.
  5. Incubate at 70°C for 15 min to terminate the reaction.
  6. Centrifuge 10 to 20 s and place the reaction at 37°C.
  7. Add 1 μl of RNase mix, mix gently but thoroughly, and incubate for 30 min at 37°C.
  8. Collect the reaction by brief centrifugation and place on ice. The procedure may be stopped at this point and the reactions stored at -20°C.


    S.N.A.P. column purification of cDNA

    1. Equilibrate the binding solution to room temperature.
    2. For each sample to be purified, equilibrate ~100 μl of sterilized, distilled water at 65°C for use in step 9.
    3. Add 120 μl of binding solution (6 M NaI) to the first strand reaction.
    4. Transfer the cDNA/NaI solution to a S.N.A.P. column. Centrifuge at 13,000 x g for 20 s.
    5. Remove the cartridge insert from the tube and transfer the flowthrough to a microcentrifuge tube. Save the solution until recovery of the cDNA is confirmed. Place the cartridge insert back into the tube.
    6. Add 0.4 ml of COLD (4°C) 1X wash buffer to the spin cartridge. Centrifuge at 13,000 x g for 20 s. Discard the flowthrough. Repeat this wash step three additional times.
    7. Wash the cartridge two times with 400 μl of COLD (4°C) 70% ethanol as described in step 6.
    8. After removing the final 70% ethanol wash from the tube, centrifuge at 13,000 x g for 1 min.
    9. Transfer the spin cartridge insert into a fresh sample recovery tube. Add 50 μl of sterilized, distilled, water (preheated to 65°C) to the spin cartridge. Centrifuge at 13,000 x g for 20 s to elute the cDNA.


    Note:    Failure to remove all the ethanol can result in poor recovery of the DNA.

    Note:        It is very important that the distilled water be at 65°C in order to maximize recovery of DNA.

    TdT tailing of cDNA

    Variable amounts of purified cDNA from the S.N.A.P. column purification may be used in the TdT-tailing reaction. Factors, such as the amount of RNA used in the first strand reaction and relative abundance of the desired mRNA, should be considered. If desired, the cDNA pool may be concentrated by lyophilization and the entire contents used in the tailing reaction. To evaluate the specificity of the subsequent amplification reaction from the oligo-dC tail, inclusion of a control reaction that omits TdT is recommended.

    1.   Add the following components to each tube and mix gently:
      ComponentVolume (μl)
      DEPC-treated water6.5
      5X tailing buffer5.0
      2 mM dCTP2.5
      S.N.A.P.-purified cDNA sample10.0
      Final Volume24.0

       
    2. Incubate for 2 to 3 min at 94°C. Chill 1 min on ice. Collect the contents of the tube by brief centrifugation and place on ice.
    3. Add 1 μl TdT, mix gently, and incubate for 10 min at 37°C.

      Final composition of the reaction:

      10 mM Tris-HCl (pH 8.4)
      25 mM KCl
      1.5 mM MgCl2
      200 μM dCTP
      cDNA
      TdT

      Note:   The 5X tailing buffer already contains MgCl2. DO NOT add it separately.

      Note:         The TdT has been optimized for 5' RACE. Increased amounts of TdT in the tailing reaction may inhibit PCR.
    4. Heat inactivate the TdT for 10 min at 65°C. Collect the contents of the reaction by brief centrifugation and place on ice.

      PCR of dC-tailed cDNA

      Tailed cDNA obtained from the preceding protocol may be amplified directly by PCR. Amplification of > 5 μl volumes of the tailing reaction requires appropriate adjustments for buffer, MgCl2, and dNTP concentrations in the PCR.
    1. Equilibrate the thermal cycler block to 94°C. In most cases, the “good start” procedure gives specific amplification products. For some target and primer sets, “hot start” has been reported to improve the specificity of the reaction (44,45).
    2. Add the following to a 0.2 or 0.5-ml thin-wall PCR tube sitting on ice:

      ComponentVolume (μl)
      sterilized, distilled water31.5
      10X PCR buffer [200 mM Tris-HCl (pH 8.4),
      500 mM KCl]      		5.0
      25 mM MgCl23.0
      10 mM dNTP mix1.0
      nested GSP2 (prepared as 10 μM solution)2.0
      Abridged Anchor Primer (10 μM)2.0
      dC-tailed cDNA5.0
      Final Volume49.5

      Note:    It is very important to have the reaction mixture ice-cold to avoid nonspecific binding and extension of primers.
    3. Add 0.5 μl of Taq DNA polymerase (5 units/μl) immediately before mixing.
    4. Mix the contents of the tube (Taq DNA polymerase is added immediately before going into the thermal cycler) and overlay with 50 to 100 μl of mineral oil (if necessary).

      Final composition of the reaction:

      20 mM Tris-HCl (pH 8.4)
      50 mM KCl
      1.5 mM MgCl2
      400 nM GSP2
      400 nM Abridged Anchor Primer
      200 μM each dATP, dCTP, dGTP, dTTP
      tailed cDNA
      2.5 units Taq DNA polymerase
    5. Transfer tubes directly from ice to the thermal cycler pre-equilibrated to the initial denaturation temperature (94°C).
    6. Perform 30 to 35 cycles of PCR. A typical cycling protocol for cDNA with ≤1 kb amplified region is:

      PAD:                                                  94°C for 1-2 min
      Cycle:
      Denaturation:                                    94°C for 0.5-1 min
      Annealing of primers:                       55°C for 0.5-1 min
      Primer extension:                             72°C for 1-2 min
      Followed by:
      Final extension:                                72°C, 5-7 min
      Indefinite hold: 5°C, until samples are removed.
    7. Analyze 5-20 μl of 5' RACE products by agarose gel electrophoresis according to standard protocols, using appropriate size standards (19). Either TAE or TBE electrophoresis buffer may be used for the procedure. The volume of the sample used for analysis will depend on the volume and thickness of the sample well. If products will be extracted for reamplification, ultraviolet (UV) visualization of ethidium bromide-stained products should be performed using either a long wavelength (356-nm) UV or 302-nm wavelength source to minimize DNA nicking.

      Nested amplification

      Often a single PCR of 25 to 35 cycles will not generate enough specific product to be detectable by ethidium bromide staining. Increasing the number of cycles performed during the PCR beyond 35 cycles may generate numerous nonspecific products and is not recommended. Similarly, high target levels may exacerbate amplification of nonspecific products during PCR and contribute to the production of a heterologous smear of PCR products. Instead, a dilution of the original PCR (0.1 to 0.05%) can be re-amplified (7) using the AUAP or UA  and a nested GSP.  If there is insufficient sequence information to design a nested GSP, re-amplification of gel purified, size-selected PCR products using the UAP, or AUAP, and original GSP is useful for enriching specific 5' RACE products or installation of dUMP-cloning sequences for UDG cloning. Nested PCR may also be conveniently conducted using a plug of agarose from the gel analysis of the initial 5' RACE reaction (63).

      1. Dilute a 5 μl aliquot of the primary PCR into 495 μl TE buffer [10 mM Tris-HCl, (pH 8.0), 1 mM EDTA].
      2. Equilibrate the thermal cycler block to 94°C.
      3. Add the following to a 0.2 or 0.5-ml thin-wall PCR tube sitting on ice.

        ComponentVolume (μl)
        sterilized, distilled water33.5
        10X PCR buffer [200 mM Tris-HCl (pH 8.4),
        500 mM KCl]        		5.0
        25 mM MgCl23.0
        10 mM dNTP mix1.0
        nested GSP (prepared as 10 μM solution)1.0
        AUAP or UAP (10 μM)1.0
        dilution of primary PCR product5.0
        Final Volume49.5

        Note:   It is very important to have the reaction mixture ice cold to avoid nonspecific binding and extension of primers.


        Note:   UDG cloning requires amplification with the UAP and appropriately designed nested GSP. Use the AUAP if using an archaeobacterial DNA polymerase or long PCR enzyme mixture.
      4. Add 0.5 μl of Taq DNA polymerase (5 units/μl) immediately before mixing.
      5. Mix the contents of the tube (Taq DNA polymerase is added immediately before going into the thermal cycler) and overlay with 50 to 100 μl of mineral oil (if necessary).

        Final composition of the reaction:

        20 mM Tris-HCl (pH 8.4)
        50 mM KCl
        1.5 mM MgCl2
        200 nM nested GSP
        200 nM UAP or AUAP
        200 μM each dATP, dCTP, dGTP, dTTP
        diluted primary PCR product
        2.5 units Taq DNA polymerase
      6. Transfer tubes directly from ice to the thermal cycler pre-equilibrated to the initial denaturation temperature.
      7. Perform 30 to 35 cycles of PCR.
      8. Analyze 5 to 20 μl of the amplified sample, using agarose gel electrophoresis, ethidium bromide staining, and the appropriate molecular size standards.

        Note:           Cycle number will depend on the amount and complexity of the target. As few as 5 cycles can be used to install dUMP-sequences for UDG cloning

 

 

Interpretation of results

Analysis of 5' RACE results:

Following PCR, products may be analyzed by agarose gel electrophoresis (1% to 2%) and ethidium bromide staining. Band intensity and size distribution of resulting products depends on the specificity of GSPs used for cDNA synthesis and PCR, the complexity and relative abundance of target cDNA, and the PCR conditions used. Amplification products may vary from a single specific band to multiple discrete products to a broad diffuse smear. Incomplete cDNA synthesis, aberrant priming of GSPs during first strand synthesis or PCR, mispriming by the anchor primer, as well as primer-dimer and other PCR artifacts may contribute to the complexity of products obtained by 5' RACE. Identification of specific product bands may be complicated by the presence of nonspecific products that are dependent on both reverse transcription and dC-tailing (36). If sequences are available for use as internal probes, it is strongly recommended that Southern blot analysis be used to identify specific product bands. Specific products can also be identified using a diagnostic restriction endonuclease digestion if the amplified cDNA sequence contains a known restriction site.

5' RACE controls:

Several controls may facilitate interpretation of results. Products that result from amplification of contaminating genomic DNA can be identified from control reactions that omit RT. An alternative approach is to include control reactions that use genomic DNA as target (6). Specificity of the anchor primer for the oligo-dC tail should be examined by performing amplification reactions with cDNA subjected to dC-tailing both in the presence and absence of TdT. Additional controls that amplify dC-tailed cDNA using each primer individually (either the Abridged Anchor Primer or GSP2) may be useful in identifying nonspecific products that result from mispriming.

Nested amplification:

5' RACE of rare messages may require additional PCR using a nested GSP and either the UAP or AUAP. Generally, a dilution of the original PCR is used as target. A nested primer is composed of sequences located 3' to the original primer (GSP2). For 5' RACE, this would be an antisense primer that anneals closer to the mRNA 5'-end. Purification of the original PCR product from primers and primer-dimer products may significantly improve the specificity and efficiency of nested amplification procedures. Ultimately, the 5' RACE procedure should produce a single prominent band on an agarose gel. This may require additional rounds of PCR using successively nested GSPs. Decisions regarding the design of a nested primer will depend on the amount of sequence information available for the target of interest and on the results of the original amplification reaction. When performing 5' RACE with a nested primer, sequences specific for downstream cloning manipulations must be designed into the nested GSP.

Troubleshooting guide

Testing the 5' RACE System using the control RNA and DNA

When using the 5' RACE System for the first time, we suggest performing an experiment using the control RNA to become familiar with the 5' RACE System procedure and to verify proper functioning of all components in the protocol, including your reagents and equipment for PCRs. The control RNA provided with the 5' RACE System is an 891-bp, in vitro transcribed RNA from the chloramphenicol acetyltransferase (CAT) gene that has been engineered to contain a 3' poly(A) tail. It may be used alone or added to your RNA preparation to test system performance in a background of heterologous nucleic acid. This is useful to test for the presence of contaminating nucleases. If desired, dilutions of the control RNA may be used to determine the sensitivity of the system or to model the abundance of the desired mRNA.

The control DNA was constructed by cloning the control 5' RACE product into pAMP1. Tailed cDNA was amplified using the 5' RACE Anchor Primer and control GSP2 containing additional UDG cloning sequences. The 4.8-kb pAMP1 5'RACE recombinant contains the oligo-dC tail sequence and may be used as a PCR positive control to verify the performance of the Abridged Anchor Primer, 5' RACE Anchor Primer, AUAP, UAP, or the control GSP3, in conjunction with the control GSP2. Alternately, it may be used to optimize PCR parameters with the Abridged Anchor Primer for your reaction conditions or thermal cycling device. Two different PCRs are used to verify system performance. Conversion of first strand cDNA and recovery of cDNA after S.N.A.P. purification are assayed by a CAT cDNAspecific PCR using the control GSP3 and GSP2. Addition of the oligo-dC tail to purified control cDNA is assayed by PCR using the Abridged Anchor Primer and control GSP2. Sequences for the control primers are presented below. The annealing sites for the control primers and resulting amplification products are shown in figure 5, panel 1. Note: The user may find it advantageous to adopt a similar RT-PCR strategy for their message and design an appropriate sense gene-specific primer (GSP3) to facilitate troubleshooting problems that may arise during 5' RACE with their message.

control GSP1 5'-TTG TAA TTC ATT AAG CAT TCT GCC-3'
control GSP2 5'-GAC ATG GAA GCC ATC ACA GAC-3'
control GSP3 5'-CGA CCG TTC AGC TGG ATA TTA C-3'

Sequences of the control primers 

Typical results for the procedure using the control RNA, both alone and in a background of 1 μg HeLa total RNA, are described next.  A distinct 711-bp 5' RACE PCR product (solid arrow) should be visible by ethidium bromide staining. Other products, generally visible as faint bands or a diffuse smear, can result from spurious priming by GSP2 or the anchor primer, incomplete cDNA synthesis, and primer-dimer artifacts. If the control RNA is used in a background of heterologous RNA, nonspecific 5' RACE products, that are dependent on both RT and TdT, may be observed. This effect is illustrated by the 310-bp HeLa-derived 5' RACE product . The presence of these nonspecific but genuine 5' RACE products is primarily a function of the specificity of the GSPs and emphasizes the need for characterization and enrichment of specific amplification products prior to cloning or sequencing.

Control first strand cDNA synthesis

The RNA template for this step is control RNA and the primer is control GSP 1. If you wish to test performance in a background of your RNA we suggest doing two first strand reactions:
(Control RNA + Control GSP 1) and (Control RNA +Control GSP 1 in the presence of your sample RNA). Adjust the volume of DEPC-treated water appropriately so that the final volume in step 1 is still 15.5 μl.

Note:   Mix and quickly centrifuge each component before use.

  1. Add the following to a 0.5-ml microcentrifuge tube:

    2. ComponentVolume (μl)
    Control GSP1 (1 μM)2.5
    control RNA1.0
    DEPC-treated water12.0
    final volume15.5


  2. Incubate the mixture 10 min at 70°C to denature RNA. Chill 1 min on ice. Collect the contents of the tube by brief centrifugation and add the following in the order given:

    ComponentVolume (μl)
    10X PCR buffer2.5
    25 mM MgCl22.5
    10 mM dNTP mix1.0
    0.1 M DTT2.5
    final volume8.5

    The combined total volume of steps 1 and 2 is 24 μl.

  3. Mix gently, and collect the reaction by brief centrifugation. Incubate for 1 min at 42°C.

  4. Add 1 μl of SuperScript II RT. Mix gently and incubate for 50 min at 42°C.

  5. Incubate at 70°C for 15 min to terminate the reaction.

  6. Centrifuge 10 to 20 s and place the reaction at 37°C.

  7. Add 1 μl of RNase Mix, mix gently, and incubate for 30 min at 37°C.

  8. Collect the reaction by brief centrifugation and place on ice.

  9. Transfer a 2-μl aliquot of the control first strand cDNA to a 1.5-ml microcentrifuge tube containing 998 μl of TE buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA]. Label the tube “A” and retain for later PCR.

  10. Purify the remainder of the control DNA using the S.N.A.P. column.

    S.N.A.P. column purification of the control cDNA


    1. Equilibrate the binding solution to room temperature.

    2. Equilibrate ~100 μl of distilled water at 65°C (for each sample to be purified) for use in step 9.

    3. Add 120 μl of binding solution (6 M NaI) to the remainder of the control first strand reaction.

    4. Transfer the cDNA/NaI solution to a S.N.A.P. column. Centrifuge at 13,000 x g for 20 s.

    5. Remove the cartridge insert from the tube and transfer the flowthrough to a microcentrifuge tube. Save the solution until recovery of the cDNA is confirmed. Place the cartridge insert back into the tube.

    6. Add 0.4 ml of COLD (4°C) 1X wash buffer to the spin cartridge. Centrifuge at 13,000 x g for 20 s. Discard the flowthrough. Repeat this wash step three additional times.

    7. Wash the cartridge two times with 400 μl of COLD (4°C) 70% ethanol as described in step 6.

      Note:   The binding solution must be at room temperature for efficient binding of the DNA.

      Note:   1X wash buffer and 70% ethanol must be prepared prior to use and used cold

    8. After removing the final 70% ethanol wash from the tube, centrifuge at 13,000 x g for 1 min.

    9. Transfer the spin cartridge insert into a fresh sample recovery tube. Add 50 μl of distilled water (preheated to 65°C) to the spin cartridge. Centrifuge at 13,000 x g for 20 s to elute the cDNA.

    10. Transfer a 5-μl aliquot of the purified control first strand cDNA to a 0.5-ml microcentrifuge tube containing 495 μl of TE buffer. Label the tube “B” and retain for later PCR

      Note:         Failure to remove all the ethanol can result in poor recovery of the DNA.

      Note:         It is very important that the distilled water be at 65°C in order to maximize recovery.

      TdT tailing of the control first strand cDNA

      A control reaction that omits TdT is included because amplification of cDNA from this control reaction can provide important data for troubleshooting RACE. For example, inclusion of a control reaction that omits TdT will help evaluate the specificity of the amplification reaction from the oligo-dC tail. Label two 0.5-ml microcentrifuge tubes “C” and “D”, respectively.

      1. In order to more sensitively test the efficiency of the tailing reaction, prepare a 100-fold dilution of the purified control cDNA. Add 1 μl of the cDNA to a 0.5-ml tube containing 99 μl of sterilized, distilled water.

      2. Add the following components to each tube:



        3. ComponentVolume (μl)
        DEPC-treated water (or sterile, distilled water)6.5
        5X Tailing Buffer5.0
        2 mM dCTP2.5
        S.N.A.P.-purified control cDNA (1:100 dilution)10.0
        final volume24


      3. Incubate for 2 to 3 min at 94°C. Chill 1 min on ice. Collect the contents of the tube by brief centrifugation and place on ice.

      4. Add 1 μl TdT to tube(s) “C”.

      5. Add 1 μl DEPC-treated water to tube(s) “D”.

      6. Gently mix the contents of each reaction and incubate for 10 min at 37°C.

      7. Heat inactivate the TdT for 10 min at 65°C. Collect the contents of the reaction by brief centrifugation and place on ice. Retain each reaction for later PCR.


      PCR of cDNA, tailed cDNA and control DNA

      The PCR of the various aliquots from each step and the tailed cDNA product(s) is done using two sets of primers in two separate PCR mixes. An additional reaction that contains 5 μl of the control DNA as target is used as a positive control to verify PCR. One set of primers will be control GSP2 and control GSP3. Specific amplification of the control first strand cDNA product, or control DNA, results in a prominent 500-bp band when analyzing products by agarose gel electrophoresis and ethidium bromide straining. The other set of primers will be control GSP2 and Abridged Anchor Primer and should result in a prominent 711-bp product (and no product from the no TdT control) or both the tailed cDNA and the control DNA. The sample templates to be amplified are:

      “A” cDNA from the first strand reaction (checks the efficiency of the cDNA synthesis reaction)
      “B” S.N.A.P. eluate sample: (checks the recovery of cDNA from S.N.A.P. column)
      “C” Tailed cDNA: (actual 5' RACE product; checks the efficiency of tailing)
      “D” No TdT control: (checks the specificity of amplification of the tailed product, and presence of inhibitors of PCR)

      Control DNA: (checks PCR with both primer sets) You may have more than one tube for each set, depending on the number of first strand reactions you have.

      1. Make two PCR mixes, one for each primer set:

        Mix I = Control GSP2 and GSP3
        Mix II = Control GSP2 and Abridged Anchor Primer

        Add the following components on ice to an appropriately sized sterile tube, e.g. 1.5-ml microcentrifuge tube. Make enough mix for n+2 reactions where n = number of template samples to be amplified. (Minimum value for n is 5: A + B +C + D + Control DNA). Volumes given in the table are for 50 μl PCRs.

        Mix I (GSP2 and GSP3)

        Note: Add the Taq DNA polymerase just before you are ready to pipet the mix into the PCR tubes containing template.

        ComponentVolume (μl) per reactionVolume (μl) 7X mix n=5Volume (μl) 12X mix n=10
        DEPC-treated water33.5234.5402.0
        10X reaction buffer5.035.060.0
        25 mM MgCl23.021.036.0
        10 mM dNTP mix1.07.012.0
        control GSP2 (10 μM)1.07.012.0
        control GSP3 (10 μM)1.07.012.0
        Taq DNA Polymerase (5 units/μl)0.53.56.0
        final volume45.0315.0540.0

        Mix II (GSP2 and Abridged Anchor Primer)

        ComponentVolume (μl) per reactionVolume (μl) 7X mix n=5Volume (μl) 12X mix n=10
        DEPC-treated water31.5220.5378.0
        10X reaction buffer5.035.060.0
        25 mM MgCl23.021.036.0
        10 mM dNTP mix1.07.012.0
        control GSP2 (10 μM)2.014.024.0
        Abridged Anchor Primer2.014.024.0
        Taq DNA Polymerase 5 units/(μl)0.53.56.0
        final volume45.0315.0540.0

         

      2. Pipet 5 μl of template samples (A,B,C,D, and control DNA) into two sets appropriately labeled thin-walled 0.2 or 0.5 ml PCR tubes. There will be one set for each PCR mix.

      3. Add 45 μl of Mix I to one set of tubes and add 45 μl of Mix II to the second set of tubes.

      4. Mix the contents of the tubes and overlay with 50 to 100 μl of mineral oil (if necessary).

      5. Equilibrate the thermal cycler block to 94°C.

      6. Transfer tubes directly from ice to the hot thermal cycler.

      7. Perform 35 cycles of PCR:

        Denature 94°C for 1 min
        Anneal 63°C for 30 s
        Extend 72°C for 2 min

      8. Incubate the reaction for 10 min at 72°C following the last cycle of PCR, then maintain reactions at 4°C.

      9. Analyze 5-20 μl of 5' RACE products by agarose gel electrophoresis according to standard protocols, using appropriate size standards (19). Either TAE or TBE electrophoresis buffer may be used for the procedure. The volume of the sample used for analysis will dependent on the volume and thickness of the sample well. If products will be extracted for reamplification, ultraviolet (UV) visualization of ethidium bromide-stained products should be performed using either a long wavelength (356-nm) UV or 302-nm wavelength source to minimize DNA nicking.

        General Troubleshooting Guidelines for the 5' RACE System

        ProblemPossible causeSuggested remedy
        No bands after electrophoretic analysis of amplified products5' RACE amplification product may
        be present but in too low a concentration
        for detection by ethidium bromide
        staining
        • Perform Southern blot analysis of amplification products using internal sequence as probe.
        • Amplify agarose gel purified material of expected size range using the AUAP or UAP, and user’s GSP2.
        • Perform nested primer amplification from either purified amplification products, dilution of original PCR (0.05%), or agarose gel plug using the UAP,or AUAP, and user’s nested GSP.
         Procedural error in first strand cDNA
        synthesis, purification of cDNA product,
        TdT tailing, or PCR
        • Use the control RNA to verify conversion of first strand product, recovery following purification and dC tailing.
        • Design sense GSP from available 5'-mRNA sequence data. Verify first strand conversion of desired message by PCR using two genespecific primers.
         Inhibitors of RT present
        • Remove inhibitors by ethanol precipitation of the mRNA preparation before the first strand reaction. Include a 70% (v/v) ethanol wash of the mRNA pellet.

        Note:         Inhibitors of RT include sodium dodecyl sulfate (SDS), EDTA, guanidinium salts, and glycerol. Inhibitors of M-MLV-RT include sodium pyrophosphate and spermidine. SuperScript™ RT is inhibited 50% by 0.0025% SDS, 1 mM EDTA , 15 mM guanidine isothiocyanate, 17% DMSO, 50% glycerol, 5% formamide, 4 μg/ml heparin and 4 mg/ml glycogen (52).

        • Test for the presence of inhibitors by mixing 1 μg of control RNA with 1 μg of sample RNA and comparing yields of first strand cDNA or by PCR of control band.
         Target mRNA has secondary structure that interferes with annealing of GSP1Redesign GSP1
         Target mRNA contains strong transcriptional pausesMaintain an elevated temperature after the annealing step and increase the temperature of first strand reaction (up to 50°C).
         No bands after electrophoretic
        analysis of amplified products        		RNase contamination
        • Perform first strand synthesis with the control RNA both alone and added to sample RNA to determine if RNase is present. Assay for control cDNA by PCR using control GSP2 and GSP3.
        • Maintain aseptic conditions to prevent RNase contamination.
        • Use placental RNase inhibitor during first strand cDNA synthesis.

        Note:         Placental RNase inhibitor requires sulfhydryl reagents for maximal RNase binding activity (19). Always add RNase inhibitor to reactions after the addition of dithiothreitol. Treatments which denature the protein, such as high temperature incubation, exposure to oxidizing conditions, or repeated freezing and thawing, can release RNases initially bound by the inhibitor. These RNases may subsequently degrade RNA preparations in downstream procedures.
         Inefficient tailing of cDNAPerform TdT time course: remove 5-μl aliquots at t = 2.5, 5, 10, and 20 min. Amplify each time point using abridged anchor primer and GSP2.
         cDNA did not tail due to strong secondary
        structure of 3'-end
        • Denature cDNA prior to tailing by incubation at 94°C for 2 to 3 min, then quick chill on ice. Perform the tailing reaction on ice for 1 h to keep 3'-end denatured and available for TdT.
        • Alternatively, use of cosolvents e.g. DMSO (≤ 20%) has been been found to increase efficiency of tailing.
         Inhibition of PCR by TdTPurify tailed cDNA by ethanol precipitation in the presence of inert carrier, i.e. glycogen, and 2.5 M NH4OAc.
         Polysaccharides and small RNAs coprecipitate with mRNAEthanol precipitate the RNA preparation; treat the pellet
         Polymerase used in PCR was from an archaeobacterium and dUMP primers were used
        • Use abridged anchor primer, or AUAP, and non-dUMP-containing GSP in PCR using eLONGase® reagents or an archaeobacterial polymerase.
        • Use Taq DNA polymerase for PCR with the UAP and dUMP-containing primers.
        Agarose gel analysis shows strong primer-dimer product, but no visible gene-specific productInefficient removal of GSP1 during S.N.A.P. procedure
        • Design GSP1 according to the guidelines above.
        • Reduce the concentration of GSP1 used in first strand synthesis.
        • Remove dC-tailed GSP1 with an additional S.N.A.P. purification after TdT tailing and second strand synthesis as described above


        Note: dCTP and the 5' RACE anchor primer may be substituted for dATP and the 3' RACE system adapter primer described in this protocol.

        • Substitute dU for dT in GSP1. Degrade dU-GSP1 following cDNA synthesis with UDG. Purify cDNA by ethanol precipitation in the presence of inert carrier, i.e. glycogen, and NH4OAc.

        Note: This technique may be useful for 5' RACE of short cDNAs that bind poorly to the S.N.A.P. column.
         Primer-dimer product between the anchor primer and GSP2Minimize nonspecific annealing of primers by initiation of PCR at an elevated temperature (75°C to 80°C).
        Unexpected bands after electrophoretic analysis of "nested" amplification productsContamination by genomic DNA Spurious priming in the PCR
        • Pretreat RNA 
        • Vary the parameters of the PCR: increase annealing temperature, decrease annealing time, reduce MgCl2 concentration, etc. (24-26, 38, 42, 43).
        • Minimize nonspecific annealing of primers by initiation of PCR at an elevated temperature (75°C to 80°C).
        • Perform PCR control amplifications as discussed above.
        Gene-specific 5' RACE products appear as a smear. Unable to isolate full length 5' RACE productRNA preparation is degraded or of poor qualityRNA preparation is degraded or of poor quality
        Absence of discrete product bands. 5' RACE products appear as a heterogeneous smearPotentially, a normal resultIdentify gene-specific products by Southern blot hybridization. Enrich for specific products by reamplification of gel-purified material or nested primer amplification
        Absence of discrete product bands. 5' RACE products
        appear as a heterogeneous smear        		High levels of poly (A)+ RNA (>1 μg) used for first strand synthesis may contribute to nonspecific products. RNA-primed, (GSP1 independent) cDNA may compete for TdT and also for anchor primer during the PCR.Reduce target level or optimize tailing conditions. Perform TdT time course: remove 5-μl aliquots at t = 2.5, 5, 10, and 20 min. Amplify each time point using abridged anchor primer and GSP2.
        5' RACE product does not correspond to known mRNA sequence. Product is dependent on TdT addition of dC-tailTarget mRNA contains strong transcriptional pausesMaintain an elevated temperature after the annealing step and increase the temperature of first strand reaction (up to 50°C).  Use cosolvents e.g., 5-10% DMSO or 10-20% glycerol compatible with the RT in first strand reaction (52) to help eliminate secondary structure while maintaining RT activity.
        5' RACE product does not correspond to full length mRNA sequence. Product is not dependent on TdT addition of dC-tailInternal mispriming by the anchor primer at C-rich sequenceDeoxyinosine-containing anchor primer may not be suitable for amplification of C-rich cDNA. Use alternate 5' RACE strategy.
        5' RACE product does not correspond to known mRNA sequence.Aberrant priming of gene-specific primers
        • Perform control amplification reactions as discussed above, 5' RACE Controls.
        • Verify identity of 5' RACE product by hybridization or diagnostic restriction endonuclease cleavage prior to cloning or sequencing.
        • Increase the specificity of GSP1 by increasing the temperature of first strand synthesis (up to 50°C).
        • Optimize PCR conditions to maximize specificity of GSP2 and anchor primer. Increase annealing temperature, decrease annealing time, decrease MgCl2 concentration, etc.
        • Design new GSP1 and/or GSP2.
        Poor cloning efficiencyInefficient ligation
        • Increase the incubation time in the ligation reaction; decrease the temperature.
        • Remove dNTPs prior to ligation.
        • Use a ligation-free cloning method such as the CloneAmp system.
         Poor restriction endonuclease digestion due to residual bound Taq DNA PolymeraseTreat PCR products with proteinase K (64).
         Fill-in of overhangs by residual Taq DNA polymeraseExtract PCR products with phenol:chloroform and purify by ethanol precipitation or treat with proteinase K (see above) before restriction endonuclease digestion.
         Clone is unstable in host cellsTry different strain of bacterial cells such as Stbl2™ competent cells.

        Minimizing RNase contamination

        Successful cDNA synthesis demands an RNase-free environment at all times, which will generally require the same level of care used to maintain aseptic conditions when working with microorganisms. Several additional guidelines should be followed:

        1. Never assume that anything is RNase-free, except sterilized pipettes, centrifuge tubes, culture tubes, or any similar labware that is explicitly stated to be sterile. Wear latex gloves for all manipulations involving RNA.

        2. Dedicate a separate set of automatic pipettes for manipulating RNA and the buffers and enzymes used to synthesize cDNA.

        3. Avoid using any recycled glassware unless it has been specifically rendered RNase-free by rinsing with 0.5 N NaOH followed by copious amounts of sterilized, distilled water. Alternatively, bake glassware at 150°C for 4 h.

        4. Microcentrifuge tubes can generally be taken from an unopened box, autoclaved, and used for all cDNA work. RNase-free microcentrifuge tubes can be purchased from several suppliers. If it is necessary to decontaminate untreated tubes, soak the tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC), rinse them with sterilized, distilled water, and autoclave them.

        5. If made with RNase-free labware, most solutions can be made from reagent-grade materials and distilled water, and then autoclaved. Prepare heat-sensitive solutions using sterilized, distilled water, and filter them to 0.2  μm using sterilized, disposable filterware.

        6. If all else fails, most aqueous buffer solutions can be treated with 0.01% (v/v) DEPC and autoclaved.

        7. Use aerosol-resistant pipet tips.

          Tm values for 5' RACE and control primers

          Oligonucleotide primer Tms can vary widely depending on the method of calculation. A comparison of Tm determinations (°C) for the 5' RACE Anchor Primer, Abridged Anchor Primer, UAP, AUAP, control GSP2, and control GSP3 using the 2(AT) + 4(GC), %GC, and nearest neighbor methods are summarized in table 1. The nearest neighbor analysis method (65) relies not only on the quantity of each base, but on the primary sequence of the DNA. Comparison of experimentally derived Tms to that predicted by the available estimates for a number of oligonucleotides has found the nearest neighbor method to be the most accurate (22,66). However, the precise reaction conditions should be considered in the calculation. Other methods are either limited by the size of the oligonucleotide for which they are valid or derived for salt and temperature conditions that are different from those for PCR (see legend, table 1, and 19,67-70).

          Table 1. Comparison of Tm Values for 5' RACE and Control Primers calculated by different methods

          Primer2(AT) + 4(GC)1%GC2NN3NN PCR4
          GI Anchor Region527260.566.6
          Anchor Primer15090.393.990.1
          Abridged Anchor Primer11890.094.492.5
          UAP9883.875.875.0
          AUAP6679.367.871.5
          GSP26475.364.569
          GSP36675.466.569.2

          Tm values (°C) for 5' RACE and control primers were calculated using OLIGO™ 5.0 according to the formulae given below. The GI Anchor Region comprises the 16-base deoxyinosine-containing sequence at the 3'-end of the Abridged Anchor or Anchor Primer. The efficiency and specificity of annealing of this sequence to the dC-tail in the early PCR cycles is critical to the overall efficiency of 5’ RACE. The disparity of Tm values obtained using different formulae highlights the importance of empirical determination of optimal annealing temperature for any given primer pair.

           
          1. The Tm of many oligonucleotides can be estimated by assigning 2°C to each A and T and 4°C to each G and C (63,64). 2(AT) + 4(GC): 2 x (numbers of A’s + number of T’s) + 4 x (number of C’s + number of G’s). This estimate was derived for hybridizations in high salt concentration (1M) and is only valid for oligonucleotides less than 18 bases in length (35).

          2. Another method for estimating the Tm is based on the percent GC in the oligonucleotide (35,65,66). The formula was developed for hybridization to DNA immobilized on a solid support using molecules under 100 bases in length in conditions of high salt. %GC: Tm = 81.5 + 16.6 log[salt+] + 0.41[%GC] -(675 / number of bases in oligonucleotide)

          3. NN: nearest neighbor Tm (19) calculated using second order hybridization kinetics, 100 pM primer 1 M Na+.

          4. NN PCR: nearest neighbor Tm (19) calculated for standard PCR conditions using first order hybridization kinetics, 200 nM primer, 50 mM Na+, 0.7 mM free Mg++ (effective [Na+] = 155.6 mM).

        TOP

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