Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs

Journal of Virological Methods 73 (1998) 1–9

Protocol

Reverse transcription polymerase chain reaction protocols forcloning small circular RNAs

Beatriz Navarro, Jose A. Daros, Ricardo Flores *

Instituto de Biologıa Molecular y Celular de Plantas (UPV-CSIC), Uni6ersidad Politecnica de Valencia, Camino de Vera 14,46022 Valencia, Spain

Accepted 16 February 1998

Abstract

A protocol is described for general application for cloning small circular RNAs which requires only minimalamounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-merprimer whose six 3%-terminal positions are totally degenerate in two consecutive reactions catalyzed by reversetranscriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with thenon-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of oneor more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is alsodescribed. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: RT-PCR protocol; Viroids; Satellite RNAs; Small circular RNAs

1. Type of research

Small circular RNAs are uncommon moleculesin nature. Some of these are the causal agents ofa series of diseases affecting animals and plantswhereas others are products of the cellularmetabolism. Within the first category are: (i)

viroids, autonomously replicating single-strandedRNAs from plants with a size below 400 nt(Diener, 1991; Flores et al., 1997), (ii) viroid-likesatellite RNAs structurally similar to viroids butfunctionally dependent on a plant helper RNAvirus (Bruening et al., 1991; Symons, 1997), (iii)the RNA of human hepatitis d virus which resem-bles viroid-like satellite RNAs in its dependenceon a virus, human hepatitis B, but has a sizeconsiderably larger, approximately 1700 nt

* Corresponding author. Tel.: +34 96 3877861; fax: +3496 3877859; e-mail: [email protected]

0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S0166-0934(98)00042-1

B. Na6arro et al. / Journal of Virological Methods 73 (1998) 1–92

(Lazinski and Taylor, 1994; Lai, 1995), and (iv)two small circular RNAs from Neurospora andcarnation which also exist in the form of a ho-mologous DNA counterpart (Saville and Collins,1990; Daros and Flores, 1995b). Included in thesecond category are some intron-derived RNAs,particularly from group I (Cech, 1990). The fre-quent occurrence of ribozymes on the small circu-lar RNAs has attracted much interest in them.

Molecular characterization of these RNAs andparticularly of viroids was carried out initially bydirect RNA sequencing (Gross et al., 1978), amaterial and time consuming methodology. Withthe development of approaches permitting cDNAamplification either in vivo by recombinant plas-mids or in vitro by the polymerase chain reaction(PCR) (Mullis and Faloona, 1987; Saiki et al.,1988), direct sequencing has been restricted inmost cases to the elucidation of a portion of theRNA sequence necessary for the synthesis of oneor more specific primers (Visvader and Symons,1985; Koltunow and Rezaian, 1988; Hernandezand Flores, 1992). This can still pose importantdifficulties when dealing with RNAs accumulatingto very low levels. Although an RT-PCR ap-proach based on the use of random hexamers(Lakshman et al., 1992) could in principle circum-vent the problem, the need of large amounts oftemplate (approximately 5 mg), as well as thepresence of single restriction sites in the cDNA tobe cloned limits its applicability.

In our laboratory we described two methodsfor cloning small circular RNAs of unknown se-quence, based on two random-PCR approaches(Froussard, 1992; Don et al., 1993), that onlyneed approximately 50 ng of purified RNA(Navarro et al., 1996). We have used them for thesuccessful cloning of a chloroplastic intron fromcherimoya (Daros and Flores, 1996), a new appleviroid (Di Serio et al., 1996) and a viroid-likeRNA from cherry (Di Serio et al., 1997). We nowdescribe a detailed account of one of the twomethods which has a more general applicationbecause of the lower background of non-specificclones obtained. Since this approach usually pro-vides only a partial information of the RNAsequence to be determined, we also include a

description on how this information can be usedfor the synthesis of a pair of specific primers forproducing full-length cDNA clones by RT-PCR.

2. Time

Viroid purification can be accomplished in 2–3days and the time required for cDNA synthesis,PCR amplification, transformation and clone se-lection is 2–3 weeks depending on the back-ground of artefactual clones. After obtaining afragment of the sequence of the small circularRNA the complete sequence can be completed inapproximately 2 weeks.

3. Materials

3.1. General considerations for purification ofsmall circular RNAs

For reasons to be discussed below this is acritical step in the case of the first method due tothe degenerate nature of the 3% terminus of theprimer used for cDNA synthesis. It is recom-mended that the small circular RNA be purifiedby two consecutive electrophoreses in polyacry-lamide gels (5%), the first under non-denaturingconditions and the second under denaturing con-ditions (Schumacher et al., 1983; Flores et al.,1985). Following this approach the small circularRNAs can be separated from the similarly sizedlinear RNAs of cellular origin which display aconsiderable faster mobility.

3.2. cDNA synthesis and PCR amplification

3.2.1. For both methodsAvian myeloblastosis virus reverse transcriptase

(AMV-RT) (25 U/m l) (Boehringer-Mannheim).Pfu DNA polymerase (2.5 U/m l) (Stratagene). PfuDNA polymerase×10 buffer (Stratagene): 200mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM(NH4)2SO4, 0.1% Triton X-100 and 0.1 mg/mlbovine serum albumin (BSA). dNTPs (10 mM)(Boehringer Mannheim).

B. Na6arro et al. / Journal of Virological Methods 73 (1998) 1–9 3

3.2.2. For method 1Klenow fragment of E. coli DNA polymerase (5

U/m l) (Boehringer Mannheim). KGB×2 buffer:200 mM potassium glutamate, 50 mM Tris, 20mM magnesium acetate, pH 7.6 with acetic acid,plus 0.1 mg/ml BSA and 1 mM mercaptoethanol.Primer I (26-mer): (5%-GACGTCCAGATCGC-GATTTCNNNNNN-3%) with NruI and EcoRIrestriction sites and six randomized positions atits 3% terminus. Primer II (20-mer): (5%-GACGTCCAGATCGCGATTTC-3%).

3.2.3. For method 2Annealing buffer×10 (100 mM Tris–HCl, pH

8.5, 200 mM KCl). AMV-RT×5 buffer (250 mMTris–HCl, pH 8.5, 150 mM KCl, 40 mM MgCl2,5 mM DTT). A pair of specific adjacent primersof opposite polarities phosphorylated previously.

3.3. Cloning

Non-denaturing electrophoresis for separationof PCR-amplified products: 5% polyacrylamidegels (13×13×0.2 cm) (acrylamide–bisacry-lamide ratio 39:1) prepared in TAE×1 (40 mMTris, 20 mM sodium acetate, 1 mM EDTA, pH7.2 with acetic acid) and run in the same buffer at70 mA constant current for 2.5 h. pUC 18 plas-mid digested with Sma I and dephosphorylated,plus T4 DNA ligase and buffer (as supplied in theReady-To-Go kit™ from Pharmacia Biotech).

3.4. Southern and colony blot hybridization

Nylon membranes (z-Probe from BioRad, orHybond-N+ from Amersham).

Pre- and hybridization solution: 50% for-mamide, 65 mM sodium phosphate pH 6.5, 60mM NaCl, 7.5 mM sodium citrate, 10 mMEDTA, 1% SDS, 0.1% polyvinylpyrrolidone, 0.1%Ficoll and 100 mg/ml of sonicated and denaturedcalf-thymus DNA (the hybridization solution issupplemented with the cDNA probe).

Washing solutions: SSC×2 (0.3 M NaCl, 0.03M trisodium citrate, pH 7.0) with 0.1% SDS, andSSC×0.1 with 0.1% SDS.

For transferring bacterial colonies: WhatmannNo. 1 paper soaked on SSC×2 containing 2%SDS, and SSC×5 with 0.1% SDS.

For synthesis of the cDNA probe: randomhexamers pd(N)6 (Pharmacia Biotech), AMV-RT(25 U/m l), dNTP mixture (10 mM dATP, 10 mMdGTP, 10 mM dTTP, 1 mM dCTP), AMV-RT×5 buffer and [a-32P]dCTP (3000 Ci/mmol)(Amersham).

3.5. Sequencing

T7 DNA polymerase sequencing kit (PharmaciaBiotech) and [35S]dCTPa (1000 Ci/mmol) (Amer-sham) for manual sequencing.

dRhodamine terminator cycle sequencing kit(Perkin-Elmer) for automatic sequencing.

3.6. Special equipment

Polytron homogenizer (Kinematica); mi-cropipettes (Eppendorf); thermocycler (MJ Re-search); EPS 3500 electrophoresis power supply(Pharmacia Biotech); 2005 Transfer Unit (LKB);ultraviolet light transilluminator (UVP); 1800Stratalinker apparatus (Stratagene); hybridizationoven (AMS Biotechnology); M730 microwaveoven (Philips); gel-camera (Polaroid); and ABIPRISM DNA Sequencer 377 (Perkin-Elmer).

4. Detailed procedure

4.1. Method 1 (random primers)

This RT-PCR approach uses for cDNA synthe-sis a primer I (26-mer) with six randomized posi-tions at its 3% terminus and a defined sequence atthe 5% moiety, and for PCR amplification primerII (20-mer) with the same sequence as primer Iexcept the six 3%-terminal randomized positions(Fig. 1).

4.1.1. RNA denaturation and primer annealingPrior to synthesis of first-strand cDNA, the

purified RNA (at least 10 ng) and primer I (100ng) in maximum volume of 6 m l of water weredenatured at 95°C for 2 min and cooled rapidlyon ice. The ratio RNA to primer I must be1:10.


Fig. 1. Flow chart diagrams of methods 1 and 2 for synthesis, PCR amplification and cloning of cDNAs from small circular RNAs.


4.1.2. Synthesis of cDNAFor first-strand cDNA synthesis 0.5 m l of

dNTPs (10 mM), 2.5 m l of KGB×2 buffer, 1 m lof AMV-RT (25 U/m l) were added to the tubecontaining the template and primer I, and thefinal reaction volume was adjusted to 10 m l withwater. The RT-catalyzed reaction was incubatedat 42°C for 30 min, and then heated to 95°C for2 min and cooled rapidly on ice for denaturing theRNA-DNA complex. For second-strand cDNAsynthesis, 1 m l of the Klenow fragment of E. coliDNA polymerase I (5 U/m l) was added and thereaction mixture was incubated at 37°C for 30min. This synthesis is primed by the excess ofprimer I used in the first step and, therefore, theobtained double-stranded cDNA fragments havethe same sequence at both termini.

4.1.3. PCR amplificationDouble-stranded cDNAs (1 m l of the previous

reaction mixture) were PCR-amplified in a finalvolume of 50 m l containing: 5 m l of PCR×10buffer, 1 m l of dNTPs (10 mM), 1 m l of thephosphorylated primer II (1 mg/m l), 1 m l of PfuDNA polymerase (2.5 U/m l) and 41 m l of H2O.The PCR profile (30 cycles) was 94°C for 40 s,60°C for 30 s and 72°C for 2 min.

4.1.4. Cloning and selection of clones of interestPCR-amplified DNAs were separated in non-

denaturing polyacrylamide gels (5%), eluted, re-covered by ethanol precipitation and cloneddirectly in the Sma I digested and dephosphory-lated pUC 18 plasmid. Selection of clones withinserts derived from the initial RNA template wasperformed by transferring the bacterial colonies tonylon filters which were placed colony face up for2 min on Whatman No. 1 paper soaked onSSC×2 containing 2% SDS, and then in a mi-crowave oven for 2.5 min at 650 W. Prior toprehybridization the filters were wetted in SSC×5 with 0.1% SDS. (Buluwela et al., 1989). Confir-mation that recombinant plasmids had the insertsof interest was carried out by Southern blot hy-bridization. Plasmids from white bacterialcolonies obtained by alkaline lysis (Sambrook etal., 1989) were digested with EcoRI and BamHI,

electrophoresed in a non-denaturing polyacry-lamide gel (5%) and electroblotted to a nylonmembrane using a 2005 transfer Unit (LKB) op-erating at 25 V for 3 h in 25 mM sodium phos-phate pH 6.5. The gel was washed previously oncein 0.2 M NaOH containing 0.5 M NaCl for 15min, twice in 250 mM sodium phosphate pH 6.5for 5 min and finally in 25 mM sodium phosphatepH 6.5 for 15 min. Nucleic acids were fixed to themembrane with a 1800 Stratalinker apparatus(Stratagene).

For synthesis of the cDNA probe specific forthe small circular RNA a purified preparation ofit (25–50 ng) was annealed with random hexam-ers (ratio 1:10) by heating at 95°C for 2 min andcooling on ice, and then mixed in a final volumeof 10 m l with 1 m l of of the dNTP mixture, 4 m l ofAMV-RT×5 buffer, 1 m l of [a-32P]dCTP (3000Ci/mmol) and 1 m l of AMV-RT (25 U/m l), andincubated at 42°C during 30 min. Pre-hybridiza-tion, hybridization (in the presence of 50% for-mamide at 55°C) and washing were carried out asreported previously (Flores, 1986).

4.1.5. SequencingRecombinant plasmids with cDNA inserts that

hybridized with the probe were sequenced withchain terminating inhibitors (Sanger et al., 1977)either manually or automatically.

4.2. Method 2 (specific primers)

When a portion of the sequence of the smallcircular RNA has been determined by the methoddescribed in Section 4.1 (or by any other alterna-tive approach), a pair of adjacent primers ofopposite polarities can be designed and used toobtain full-length cDNA clones by RT-PCR (Fig.1).

4.2.1. RNA denaturation and primer annealingThe purified small circular RNA (approxi-

mately 10 ng) and 100 ng of the complementaryprimer (preferably at least of 20-mer) were dis-solved in a maximum volume 14 m l of water,heated at 95°C for 2 min and cooled rapidly onice.


4.2.2. cDNA synthesis, cloning and sequencingFor first-strand cDNA synthesis, 1 m l of

dNTPs (10 mM), 4 m l of RT×5 buffer, 1 m l ofAMV-RT (25 U/m l) were added to the tube con-taining the template and primer, and the finalreaction volume was adjusted to 20 m l with wa-ter and incubated at 42°C for 1 h. PCR amplifi-cations were carried out as indicated above (seeSection 4.1.3.) but using, instead of primer II, 0.5mg each of the two specific primers previouslyphosphorylated. PCR-amplified DNAs of the ex-pected full-size were eluted, cloned, and se-quenced as indicated above.

5. Results

The conditions described in the detailed proce-dure of Section 4.1. were determined using av-ocado sunblotch viroid (ASBVd) of a knownsequence of 247 nt, in order to better definethese conditions and particularly the smallestamount of RNA template required, the optimaltemplate to primer ratio, and the PCR cyclingprofile (Navarro et al., 1996). This protocol hasbeen subsequently used to clone a viroid and aviroid-like RNA from apple and cherry, respec-tively (Di Serio et al., 1996, 1997) whose se-quences were unknown.

The detailed procedure of Section 4.2., or vari-ations thereof, has been applied to obtain full-length cDNA clones of small circular RNAsfrom which a portion of their sequences wereknown (Puchta and Sanger, 1989; Hadidi andYang, 1990; McInnes and Symons, 1991; Darosand Flores, 1995a). We have found occasionallyaccompanying the expected full-length product,other PCR-amplified DNAs with higher andsmaller sizes which we presume are due to thegeneration of tandem repeated copies by the re-verse transcriptase when acting on circular tem-plates in the first case, and to non-specificannealing of the primers in the second case. In-creasing the size of the primers, which permits aparallel increase of the annealing temperatureduring the PCR reaction, helps to ameliorate thesecond situation. After establishing the complete

sequence of the cDNA it is recommended to usethis information for the synthesis of a new pairof adjacent and complementary primers in orderto obtain a second cDNA to confirm the initialsequence and to evaluate the heterogeneitypresent in the fragment covered by the first pairof primers.

6. Discussion

6.1. Purity of the RNA template (method 1)

The circular RNA used for cloning purposesmust be as pure as possible. Since the primerused in the cDNA synthesis reactions has a 3%tail of random hexamers, any contaminatingRNA present in the nucleic acid preparation, forexample fragments of ribosomal RNAs, can be asubstrate and produce a significant backgroundof non-specific products. On the other hand, thecircular RNA used for synthesis of the radioac-tive probe must be also essentially free of accom-panying cellular RNAs. For this purpose it isrecommended to purify the small circular RNAby two consecutive electrophoretic steps in poly-acrylamide gels (5%) under non-denaturing anddenaturing conditions, respectively (Schumacheret al., 1983; Flores et al., 1985). An additionaldenaturing electrophoresis helps to remove con-taminants. However, total elimination of impuri-ties is impossible because even purified reversetranscriptase preparations may contain variableamounts of nucleic acids from the virus or thehost cell. Therefore, it is necessary to select thespecific clones by colony or Southern blot hy-bridization.

6.2. Template to primer ratio (method 1)

The proportion of specific cDNA clones ob-tained for a ratio template to primer of 1:10 wasapproximately 65%, but this proportion de-creased considerably when the ratio was in-creased to 1:100 (Navarro et al., 1996).Therefore, this is a parameter that must be care-fully evaluated.


6.3. Primer design (method 2)

The specific cDNA inserts obtained by the firstmethod were typically shorter-than-full-length, asit could be anticipated since synthesis of second-strand cDNA is expected in most cases to startfrom internal positions of the first-strand cDNA(Fig. 1). However, the sequence information ob-tained from the partial-length inserts is sufficientto produce a pair of specific and adjacent primersof opposite polarities to obtain full-length cDNAsby the second method. There are some importantaspects in the design of these primers: (i) their sizemust be at least 20-mer in order to promote acorrect annealing reducing in this way non-spe-cific amplifications, (ii) they should preferablycover a non-structured region of the most stablesecondary structure to facilitate the opening ofthis region and annealing, and (iii) they must havea reduced potential to form primers-dimers. Thereare several useful programs to optimize primerdesign; in our hands the OLIGO program hasworked well.

6.4. Re6erse transcription (method 2)

The efficiency of this step can be improved byusing an annealing buffer (10 mM Tris–HCl, pH8.5, 20 mM KCl) instead of water in Section 4.2.1.of the detailed protocol. This buffer does notcontain Mg2+ in order to prevent the self-cleav-age of small circular RNA harboring hammer-head, hairpin or other ribozymatic structureswhich require this cation as a cofactor. Self-cleav-age of the circular RNA generates linearmolecules that can not be amplified subsequently.

6.5. Source of thermostable DNA polymerase andcontaminations (both methods)

The two detailed protocols described above arebased on previous reports in which Taq DNApolymerase was used in PCR amplification. How-ever, in these cases in which maximum fidelity isdesired, a thermostable DNA polymerase en-dowed with a proof-reading activity should beused. Pfu DNA polymerase is an appropriatechoice because of its very low error rate (Cline etal., 1996).

Contamination is a potential risk in any PCR-based approach. To monitor this source of arti-facts, all amplification experiments should beperformed along with a control containing all theingredients of the PCR mixture except the cDNAtemplate. It is also recommended to run a secondtype of control in which the PCR mixture issupplemented with the products resulting from anRT reaction mixture without added RNA tem-plate; the PCR products obtained from this con-trol show the magnitude of the amplification dueto nucleic acids impurities accompanying thesmall circular RNA.

7. Essential literature references

The first method described above is a moredetailed and revised version of that reported pre-viously (Navarro et al., 1996) based on a random-PCR approach proposed to construct wholecDNA libraries from low amounts of RNA(Froussard, 1992). The second method, based onthe use of specific primers, is an adaptation ofpreviously published protocols (Puchta andSanger, 1989; Hadidi and Yang, 1990; McInnesand Symons, 1991; Daros and Flores, 1995a).

8. Quick procedure

8.1. Method 1

1. Mix 10 ng of purified RNA and 100 ng ofprimer in a volume of 6 m l of water, heat at95°C for 1.5 min and cool on ice.

2. Add 0.5 m l of 10 mM dNTPs, 2.5 m l ofKGB×2 buffer, 1 m l of AMV-RT (25 U/m l),adjust to 10 m l with water, incubate at 42°Cfor 30 min and chill on ice. Heat at 95°C for 2min and cool on ice, add 1 m l of Klenow DNApolymerase I (5 U/m l) and incubate at 37°Cfor 30 min.

3. Mix 1 m l of the previous reaction mixture with5 m l of PCR×10 buffer, 1 m l of 10 mMdNTPs, 1 m l of the phosphorylated primer II(1 mg/m l), 1 m l of Pfu DNA polymerase (2.5U/m l), and 41 m l of H2O. PCR amplify (30


cycles) at 94°C for 40 s, 60°C for 30 s and 72°Cfor 2 min.

4. Separate PCR products by polyacrylamide gelelectrophoresis, elute the most abundantDNAs as revealed by ethidium bromide stain-ing and clone them in SmaI digested anddephosphorylated pUC 18.

5. Use a cDNA probe specific for the small circu-lar RNA prepared by reverse transcriptionwith random hexamers to select by colony blothybridization the clones with the inserts ofinterest, and confirm their nature by Southernhybridization of EcoRI–BamHI digested plas-mids.

6. Sequence the inserts.

8.2. Method 2

1. Mix 10 ng of purified RNA and 100 ng of thecomplementary primer in a volume of 14 m l ofwater, heat at 95°C for 1.5 min and cool on ice.

2. Add 4 m l of RT×5 buffer, 1 m l of 10 mMdNTPs, 1 m l of RT-AMV (25 U/m l) and incu-bate at 42°C for 1 h. Amplify by PCR as in thethird paragraph of Section 8.1. but substitutingprimer II by the two specific phosphorylatedprimers. Clone and sequence the full-lengthcDNA according to standard techniques.

Acknowledgements

We are grateful for financial support from theDireccion General de Investigacion Cientıfica yTecnica de Espana (grants PB92-0038 and PB95-0139 to R.F.) and for fellowships from the Minis-terio de Educacion y Ciencia (to B.N. and J.A.D).The help of Drs C. Hernandez and Mary Schaadin preparing Fig. 1 and reviewing the English text,respectively, is also acknowledged.

References

Bruening, G., Feldstein, P.A., Buzayan, J.M., van Tol, H.,deBear, J., Gough, G.R., Gilham, P.T., Eckstein, F., 1991.Satellite tobacco ringspot virus RNA: self-cleavage andligation reactions in replication. In Maramorosh, K. (Ed.),

Viroids and Satellites: Molecular Parasites in the Frontiersof Life. CRC Press, Boca Raton, pp. 141–158.

Buluwela, L., Forster, A., Boehm, T., Rabbits, T.H., 1989. Arapid procedure for colony screening using nylon filters.Nucleic Acids Res. 17, 452.

Cech, T.R., 1990. Self-splicing of group I introns. Annu. Rev.Biochem. 59, 543–568.

Cline, J., Braman, J.C., Hogrefe, H.H., 1996. PCR fidelity ofPfu DNA polymerase and other thermostable DNA poly-merases. Nucleic Acids Res. 24, 3546–3551.

Daros, J.A., Flores, R., 1995a. Characterization of multiplecircular RNAs derived from a plant viroid-like RNA bysequence deletions and duplications. RNA 1, 734–744.

Daros, J.A., Flores, R., 1995b. Identification of a retroviroid-like element from plants. Proc. Natl. Acad. Sci. USA 92,6856–6860.

Daros, J.A., Flores, R., 1996. A group I plant intron accumu-lates as circular RNA forms with extensive 5% deletions invivo. RNA 2, 928–936.

Di Serio, F., Aparicio, F., Alioto, D., Ragozzino, A., Flores,R., 1996. Identification and molecular properties of a 306nucleotide viroid associated with apple dimple fruit dis-ease. J. Gen. Virol. 77, 2833–2837.

Di Serio, F., Daros, J.A., Ragozzino, A., Flores, R., 1997. A451-nucleotide circular RNA from cherry with hammer-head ribozymes in its strands of both polarities. J. Virol.71, 6603–6610.

Diener, T.O., 1991. Subviral pathogens of plants: viroids andviroidlike satellite RNAs. FASEB J. 5, 2808–2813.

Don, R.H., Cox, P.T., Mattick, J.S., 1993. A ‘one tube reac-tion’ for synthesis and amplification of total cDNA fromsmall number of cells. Nucleic Acids Res. 21, 783.

Flores, R., 1986. Detection of citrus exocortis viroid in crudeextracts by dot-blot hybridization: conditions for reducingspurious hybridization results and for enhancing the sensi-tivity of the technique. J. Virol. Meth. 13, 161–169.

Flores, R., Duran-Vila, N., Pallas, V., Semancik, J.S., 1985.Detection of viroid and viroid-like RNAs from grapevine.J. Gen. Virol. 66, 2095–2102.

Flores, R., Di Serio, F., Hernandez, C., 1997. Viroids: thenon-coding genomes. Semin. Virol. 8, 65–73.

Froussard, P., 1992. A random-PCR method (rPCR) to con-struct whole cDNA library from low amounts of RNA.Nucleic Acids Res. 20, 2900.

Gross, H.J., Domdey, H., Lossow, C.H., Jank, P., Raba, M.,Alberty, H., Sanger, H.L., 1978. Nucleotide sequence andsecondary structure of potato spindle tuber viroid. Nature273, 203–208.

Hadidi, A., Yang, X., 1990. Detection of pome fruit viroids byenzymatic cDNA amplification. J. Virol. Meth. 30, 261–270.

Hernandez, C., Flores, R., 1992. Plus and minus RNAs ofpeach latent mosaic viroid self cleave in vitro via hammer-head structures. Proc. Natl. Acad. Sci. USA 89, 3711–3715.

Koltunow, A.M., Rezaian, M.A., 1988. Grapevine yellowspeckle viroid: structural features of a new viroid group.Nucleic Acids Res. 16, 849–864.


Lai, M.M.C., 1995. The molecular biology of hepatitis deltavirus. Annu. Rev. Biochem. 64, 259–286.

Lakshman, D.K., Tavantzis, S.M., Boucher, A., Singh, R.P.,1992. A rapid and versatile method for cloning viroids andother circular plant RNAs. Anal. Biochem. 203, 269–273.

Lazinski, D.W., Taylor, J.M., 1994. Recent developments inhepatitits delta virus research. Adv. Virus Res. 43, 187–231.

McInnes, J.L., Symons, R.H., 1991. Comparative structure ofviroids and their rapid detection using radioactive andnonradioactive nucleic acid probes In Maramorosh, K.(Ed.), Viroids and Satellites: Molecular Parasites in theFrontiers of Life. CRC Press, Boca Raton, pp. 21–58.

Mullis, K.B., Faloona, F.A., 1987. Specific synthesis of DNAin vitro via a polymerase–catalyzed chain reaction. Meth-ods Enzymol. 155, 335–350.

Navarro, B., Daros, J.A., Flores, R., 1996. A general strategyfor cloning viroids and other small circular RNAs that usesminimal amounts of template and does not require priorknowledge of its sequence. J. Virol. Methods 56, 59–66.

Puchta, H., Sanger, H.L., 1989. Sequence analysis of minuteamounts of viroid RNA using the polymerase chain reac-tion (PCR). Arch. Virol. 106, 335–340.

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi,R., Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a ther-mostable DNA polymerase. Science 239, 487–491.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. MolecularCloning a Laboratory Manual, 2nd ed. Cold Spring Har-bor Laboratory Press, Cold Spring Harbor.

Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencingwith chain terminating inhibitors. Proc. Natl. Acad. Sci.USA 74, 5463–5467.

Saville, B.J., Collins, R.A., 1990. A site-specific self-cleavagereaction performed by a novel RNA in Neurospora mito-chondria. Cell 61, 685–696.

Schumacher, J., Randles, J.W., Riesner, D., 1983. A twodimensional electrophoretic technique for the detection ofcircular viroids and virusoids. Anal. Biochem. 135, 288–295.

Symons, R.H., 1997. Plant pathogenic RNAs and RNA catal-ysis. Nucleic Acids Res. 20, 2683–2689.

Visvader, J.E., Symons, R.H., 1985. Eleven new sequencevariants of citrus exocortis viroid and the correlation ofsequence with pathogenicity. Nucleic Acids Res. 13, 2907–2920.

.

Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs

Documents

Transcript of Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs