Methods

3.1. Purification Isolation of RNA free of contaminants and RNases may be the of Viral RNA most critical step for the amplification of dsDNA copies of full-

length RNAs representing each of the eight vRNAs that compose the genome of influenza A virus. There are many RNA isolation kits and "home-brews" available to choose from, and most of these can be successfully employed for the isolation of high-quality RNA.

In fact, it is the laboratory researchers' attention to detail and maintenance of an RNase-free environment that is critical for RTPCR of genomic-length fragments. That being said, we find that the use of QIAGEN products for low- to high-throughput RNA extraction work well, and we recommend using Qiamp vRNA extraction kit or RNeasy RNA extraction kit because they offer the most reproducible results for multiple researchers that have different sources of virus-containing starting material. This section focuses on the RNeasy extraction procedure as it is very simple and works well for the isolation of viral RNA from tissue culture supernatants, allantoic fluid, or primary swab specimens, and it can be used for low-, medium-, and high-throughput procedures (see Note 4).

3.1.1. Important Factors for RNA Purification

Avoid Contamination

Most downstream techniques employ very sensitive PCR procedures so extreme care should be used to avoid DNA contamination of the tubes, solutions, etc.

If possible work in a dedicated "clean-BSC" within a clean room in which nucleic acid isolation is conducted, but the use of PCR amplification products, plasmids, and/or virus amplification is avoided.

Clean the bench area and replace any bench liners in preparation for noninfectious work. (Recommend 1-10% bleach, followed by 70% ETOH).

RNases are stabile, ubiquitous, and on our skin so use gloves (recommend nitrile).

Use RNase/DNase-free tubes and handle them as little as possible.

Label all RNA isolation and plasticware containers so all members of lab are aware to keep these items free of contaminating RNases or nucleic acids (e.g., RNA only, recommend storing tubes and other plasticware in the original containers that each user individually controls).

Pour tubes from bag or beaker onto saran wrap, pickup, close lid, label, and rack.

Use filter tips for pipetting.

Use dedicated tube openers to open microcentrifuge tubes, rather than hands.

Other Points • Biosafety. Influenza viruses are human and animal pathogens for Consideration that are transmitted via aerosols and direct contact, so always work with infectious virus in a certified biosafety cabinet using appropriate personal protective equipment and precautions.

• Check off protocol steps when they are completed.

• Always setup an extraction negative control(s) to identify contamination.

Read through RNA cleanup protocol, pg 56 RNeasy mini Handbook (version 04/2006). Follow safety guidelines for reagents and solutions (Buffer RLT contains high concentration of guanidine salt).

3.1.2. RNA Purification 1. Aliquot enough RLT for the total number ofRNA purifica-

Procedure tions being done into a 50-ml conical tube (e.g., 3.6 ml for 9

samples and 1 negative control).

2. Add 350 ml of RLT to each labeled individual snap-cap tube.

3. Add 100 ml ofvirus-containing allantoic fluid, culture supernatant, or swab specimen and pipette five times to mix (If you have less than 100 ml, adjust the volume up to a final of 100 ml using RNase-free ddH2O).

4. Cap and mix well and incubate 5-10 min at room temperature.

5. Setup RNeasy Mini spin columns in 2-ml collection tubes (supplied) in a stable rack (make sure to label the column appropriately).

6. Add 250 ml molecular biology grade ethanol (100%) to the diluted RNA, and mix well by pipetting eight times, and transfer the sample (~700 ml) immediately to the spin columns placed in a 2-ml collection tube (setup in step 5).

7. Close the lid gently and centrifuge for 15 s at 8,000 xg. Discard the flow-through and return spin column to the same collection tube. (Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Initial centrifugation should be done at(20°C) and elution should be done at 4°C).

8. Add 500 ml Buffer RPE to the spin column. Close the lid and centrifuge for 15 s at 8,000 xg. Discard the flow-through.

9. Add 500 ml Buffer RPE to the spin column. Close the lid and centrifuge for 2 min at 8,000 x g.

10. Place the RNeasy spin column into a new 2-ml collection tube (supplied in kit), and discard the old collection tube with the flow-through.

11. Close the lid and centrifuge at maximum speed (12,00016,000 xg) for 1 min.

12. Carefully remove the column after centrifugation and place the RNeasy spin column into a new 1.5-ml collection tube (supplied in kit).

13. Add 30 ml RNase-free water to the center of the spin column membrane. Close the lid gently and centrifuge for 1 min at 12,000-16,000 x g at 20-25°C to elute the vRNA. (The caps of the tubes are likely to break off during this step, so be prepared to transfer the eluted RNA to a new well-labeled tube).

3.2. InfluenzaA Virus Genomic Amplification by Multi-segment RT-PCR

3.2.1. Important Factors

3.2.2. M-RTPCR Procedure

14. Keep these tubes at 4°C or on ice while working in the lab and store the RNA at -80°C for fUture use. (The RNA concentration is usually so low that the spectrophotometer is not useful for determining the concentration).

This section describes the use of M-RTPCR to simultaneously amplify the complete genome using 1 set of three oligonucleotide primers (7). There are two sets of primers (3 primers/set) that have been optimized for efficient M-RTPCR. These 5'-tailed primers are complimentary to the conserved 5'- and 3' termini of each vRNA segment, which base pair to form the promoters for influenza virus RNA transcription and replication.

Avoiding contamination

• As discussed for RNA purification, PCR procedures are very sensitive and once a lab begins to use M-RTPCR, amplicons and clones of all influenza A virus vRNAs are being processed throughout the workspace. Therefore, unidirectional workflow should be strongly considered. Keep reaction setup areas of the laboratory separate from analysis and cloning areas.

• Virtually all other considerations listed for RNA purification are also applicable for RTPCR.

• Keep primer stocks and reagents well maintained (proper storage etc.) and free from contamination.

1. Isolate the influenza RNA from 100 ml of allantoic fluid, tissue culture supernatant, or swab material as described in Subheading 3.1, or thaw on ice if stored at -80 (see Note 5).

2. Thaw either the MBTuni or the Uni/Inf primer set (described in Subheadings 2 and 4).

3. Thaw Superscript III HF RT-PCR reagents and mix by vortexing (except for the enzyme), then place on ice.

4. Determine the volume of RNA (usually use 2.5 or 5 ml) to be used per reaction, the volume of DEPC-treated water to be added to the master mix, and the volume of master mix to be added to each tube from the total reaction volume (typically a 25 ml reaction is sufficient for cloning and one should double the volumes below for 50 ml reactions for subsequent sequencing).

Typical reaction planning example for a total reaction volume of25.0 ml:

If the volume of RNA per M-RTPCR reaction tube is 2.5 ml

Then volume of DEPC-treated ddH2O added to Master Mix should be 8.5 ml

And the volume of Master Mix (per tube) should be 22.5 ml

5. Make up the Master Mix on ice. Add H2O first and the enzyme last.

Typical master mix example using Uni/Inf primer set (see Note 6):

Per reaction

Total volume for 25 reactions

8.5 ml DEPC-treated ddH2O 12.5 ml 2x RT-PCR buffer 0.20 ml, 10 mM Uni-12/Inf1 0.30 ml, 10 mM Uni-12/Inf3 0.5 ml, 10 mM Uni-13/Inf1 0.5 ml RT/HiFi enzyme mix

212.5 ml DEPC ddH2O

312.5 ml 2x RT-PCRbuffer

12.5 ml RT/HiFi enzyme mix

6. Add 22.5 ml of Master Mix to 24 PCR tubes (0.2 ml) at 4°C (see Note 7).

7. Add 2.5 ml of RNA template (including negative RNA purification control) or ddH2 O (PCR negative control) to tubes (see Note 8).

Make sure to record which tube # corresponds to each sample in your notebook.

8. Place reaction tubes into a temperature cycler that is paused at 42°C (warm start).

9. Select the 3 stage-cycling parameters:

42°C/60 min; 94°C/2 min, then 5 cycles of (94°C/30 s; 44°C/30 s; 68°C/3 min), followed by 23-31 cycles of (94°C/30 s; 57°C/30 s; 68°C/3 min) and then hold at 4°C (see Note 9).

10. Analyze 5 ml of the M-RTPCR reactions by 0.8% agarose gel electrophoresis. An example of the results obtained from optimal (A) and typical (B) M-RTPCR amplifications are shown in Fig. 1. Differences in migration of the HA and NA segments are sometimes easily detected prior to sequencing [compare lanes 1-4 (H1N1) with lanes 5-10 (H7N2)] (Fig. 1b). The polymerase vRNAs are very similar in size (PB2 and PB1 are 2.3 kb and PA is 2.2 kb) so they migrate together.

11. Store amplicons at -20°C.

This procedure typically yields 50-80 ng/ml of DNA and this is related to the amount and quality of the template RNA.

3.3. Purification The M-RTPCR (and standard RTPCR) amplicons can be used of Amplicons for directly in various applications such as enzymatic digestion, subtyping

Nucleotide Sequencing with specific primers, and can be directly cloned (particularly if you or Cloning are using TOPO TA described below). However, to obtain optimal sequencing quality and cloning efficiency, the amplicons should be purified using commercially available PCR cleanup columns such as the QIAGEN QIAquick PCR purification kit used in this protocol

Fig. 1. M-RTPCR amplification of human and avian influenza A viruses. The viral genomes of a 2009 pandemic H1N1 virus (a) and a series of hemagglutination-positive specimens isolated swabs collected from live bird markets in New York (b) were amplified by M-RTPCR with the Uni/Inf primer set, and 5 ml from each of the reactions was analyzed by agarose gel electrophoresis (negative image of the ethidium bromide-stained gels are shown). The vRNA segment corresponding to the amplicon is listed to the left of panel A. The amplicons in panel B lanes 1-10 were subsequently sequenced and the subtype of viruses corresponding to lanes 1-4 was determined to be H1N1 and lanes 5-10 were determined to be H7N2 (note the slight shift in the migration pattern of the HA and NA vRNA amplicons). L, 1 kb + DNA ladder (Invitrogen); P, positive control for M-RTPCR (A/USSR/77, H1N1); N1, RNA purification negative control; and N2, M-RTPCR ddH2O template control.

Fig. 1. M-RTPCR amplification of human and avian influenza A viruses. The viral genomes of a 2009 pandemic H1N1 virus (a) and a series of hemagglutination-positive specimens isolated swabs collected from live bird markets in New York (b) were amplified by M-RTPCR with the Uni/Inf primer set, and 5 ml from each of the reactions was analyzed by agarose gel electrophoresis (negative image of the ethidium bromide-stained gels are shown). The vRNA segment corresponding to the amplicon is listed to the left of panel A. The amplicons in panel B lanes 1-10 were subsequently sequenced and the subtype of viruses corresponding to lanes 1-4 was determined to be H1N1 and lanes 5-10 were determined to be H7N2 (note the slight shift in the migration pattern of the HA and NA vRNA amplicons). L, 1 kb + DNA ladder (Invitrogen); P, positive control for M-RTPCR (A/USSR/77, H1N1); N1, RNA purification negative control; and N2, M-RTPCR ddH2O template control.

(see Note 10). Due to the high specificity of the M-RTPCR amplification, purification from the agarose gel is generally not necessary and will result in significant loss of the amplicons.

3.3.1. DNA Amplicon Before starting, read through the QIAGEN MinElute Handbook

Purification Procedure (03/2008) and be sure that all buffers are at the appropriate working concentrations.

1. Add 24 ml of 100% molecular biology grade ethanol into the buffer PE (concentrate) bottle to obtain 30 ml working solution of buffer PE.

2. Add 5 volumes (100 ml) of buffer PB to 1 volume (20 ml) of the M-RTPCR products and mix. Add 1 ml of 3 M sodium acetate (pH 5) and mix.

3. Place a QIAquick spin column in a 2-ml collection tube (provided).

4. Apply the sample to the column and centrifuge for 1 min at 16,000 x g.

5. Discard flow-through and place the column back into the same 2-ml tube.

6. Add 0.75 ml buffer PE into the column, let it stand for 2 min, and centrifuge for 1 min at 16,000 x g.

7. Discard flow-through and place the column back in the same tube. Centrifuge the column for an additional 1 min at 16,000 x g.

8. Make sure that no liquid is on the side or bottom of the column and place it in a clean 1.5-ml microcentrifuge tube.

9. Add 30 ml buffer EB to the center of the column and ensure that it completely covers the membrane, let the column stand for 1 min and centrifuge for 1 min at 16,000 xg (see Note 11).

10. Measure the concentration of the purified DNA with spectrophotometer and/or run 2 ml on an agarose gel with known standards to estimate/confirm the DNA concentration.

11. Use for subsequent procedures or store at -20°C.

Sanger-based nucleotide sequencing reactions are routinely carried out by institutional cores or commercial services. Therefore, this section focuses only on the factors important for initial reaction setup and the sequencing reaction method is not discussed in detail. For our Sanger approach, outlined below, the DNA is sequenced on an Applied Biosystems (ABI, by Life technologies, Carlsbad, California) 3730xl or ABI 3130xl sequencer using BigDye v3.1 Terminator Kits, and the results are analyzed with ABI Sequence Analysis 5.2 using the KB basecaller (see Note 12). For the typical automated DNA sequencing approaches used to be successful, two factors are crucial (1) the template should be of high purity and (2) the template must be accurately quantified. Failure to meet either of these criteria may result in poor or no useful sequence data.

The M-RTPCR amplicons can be sequenced directly after purification in Subheading 3.4, using virus-specific primers. Ideally, a combination of spectrophotometry and agarose gel electrophoresis of the purified amplicons will give the best estimation of the amount of the DNA to use for each sequencing reaction. However, we found that the following conditions work reasonably well, given the primers match the template.

3.4.1. Sanger-Sequencing 1. Amplify the genome using M-RTPCR (recommend 50 ml reac-Procedure (See Note 13) tions) or any region of the genome using strain-specific primers and purify them as described above (Subheading 3.4).

2. To sequence the PB2, PB1, and PA segments, mix 5 ml (~200 ng) of purified M-RTPCR amplicons with 3.3 pmol of virus sequence-specific primer for one sequencing reaction.

3. To sequence the HA, NP, NA, M, and NS, mix 2 ml (~80-100 ng) of purified M-RTPCR amplicons with 3.3 pmol of sequence-specific primers for one sequencing reaction.

4. If you are sequencing small regions of amplified DNA, a good rule of thumb is to multiply the length of the product by 0.1 to determine the number of nanogram to mix with 3.3 pmol of primer.

3.4. Sanger Sequencing of M-RTPCR Amplicons

3.4.2. Sequencing Ampllcons from Uncharacterized Influenza A Virus Positive Specimens

3.5. Recombination-Based Cloning of M-RTPCR Amplicons into Reverse-Genetics Plasmids

3.5.1. Rational and Design of Recombination-Based Bidirectional Reverse-Genetics Plasmids pBZA15 and pBZ61A18 for Use In Eight-Plasmid-Based Rescue of Influenza A Viruses (See Note 14)

M-RTPCR will amplify any influenza A virus and it can be used on specimens for which sequence information is not available. Two approaches can be easily used to initially determine the probable virus lineage and subtype so that strain-specific primers can be identified for subsequent sequencing.

1. Sequence the M-RTPCR products using a conserved M-segment primer (M-217, 5' -TCACGCTCACCGTGCCC AG-3') and analyze the results using Blast (10).

2. Gel purify each segment using commercially available kits and use one of the M-RTPCR primers (e.g., MBTuni-12) per sequencing reaction to partially sequence each gene (for small gene segments such as NS and M, the use of both amplification primers in two separate reactions will provide complete coverage of the gene).

The strict requirement for precise initiation and termination of influenza vRNA-like transcripts that is required for reverse genetics dramatically limits the restriction endonucleases available for cloning DNA copies of influenza vRNAs into reverse-genetics plasmids. Thus, cloning techniques independent of restriction sequence and ligation significantly simplify and accelerate this process. Therefore, we developed modified reverse-genetics plasmids that can be used to directly incorporate M-RTPCR amplicons (or more traditional vRNA amplicons) based on recombination between identical sequences in the termini of the M-RTPCR amplicons and the linearized reverse-genetics plasmids (7).

We modified a bidirectional reverse-genetics plasmid pDZ (9) by inserting a linker of 22 bp, to introduce an In-Fusion™ cloning site between the existing RNA polymerase I promoter and terminator, creating pBZ61A15 (Fig. 2). Some influenza A virus vRNAs have a cytosine substitution at position 4 of the 3' terminus; therefore, pBZ61A18, which has a guanosine substituted for the adenosine in the fourth position of the complement of the 3'-terminal promoter element of influenza A virus was also created. These reverse-genetics plasmids were designed for cloning dsDNA copies of any influenza A virus vRNA segment using recombination between short regions (15-19 nucleotides) of identity between the plasmid and influenza A virus amplicons using commercially available enzymes. Ligase-independent recombination-based cloning can be accomplished using "home-brew" approaches (11); however, the In-Fusion kit (Clontech) is recommended because of its efficiency and reproducible results. Amplicons of each of the eight vRNA segments that are present after M-RTPCR can be cloned simultaneously into the PifI-linearized pBZ61A15 or pBZ61A18 plasmids in a single reaction by using In-Fusion cloning (see Note 15). Alternatively, any amplicon of an influenza A vRNA segment, which is appropriately

Recombination-based Reverse-genetics Plasmid

M-RTPCR Amplicons pPol I |~

22 bp insert ,

Pst 1

Uni13/Inf1

-CCGGGTTATTAGTAGAAACTGCAIGCTTTTGCTCCCCCC--GGCCCAATCATCATCTTTGIACGTCGAAAACGAGGGGGG-

Psfldigestion pPol I |~

22 bp insert ,

Pst 1

-CCGGGTTATTAGTAGAAACTGCAIGCTTTTGCTCCCCCC--GGCCCAATCATCATCTTTGIACGTCGAAAACGAGGGGGG-

Uni13 Uni12 | 5'CCGGGTTATTAGTAGAAACAAGG---CCTGCTTTTGCTCCCCCC-3 '

3'GGCCCAATAATCATCTTTGTTCC---GGACGAAAACGAGGGGGG-5'

3'-exonuclease activity | (In-Fusion)

3'-exonuclease activity j (In-Fusion)

Uni12-Inf1

3'-exonuclease activity | (In-Fusion)

5'CCGGGTTATTAGTAGAAACAAGG---3'

'---GGACGAAAACGAGGGGGG-5'

Recombination (In-Fusion)

-GGCCCAATCATCATCTTTG

Fig. 2. Recombination-based cloning of M-RTPCR amplicons into reverse-genetics plasmids. Schematic diagram of the procedure used to clone M-RTPCR amplicons into our recombination-based reverse-genetics plasmid pBZ61A15. The M-RTPCR amplicons (1 segment shown) contain a dsDNA copy of a vRNA segment flanked by ten (5') or six (3') bp that are derived from the 5'-tails incorporated into the M-RTPCR primers (Uni13/Inf1 and Uni12/Inf1). The 13-conserved nucleotides at the 5' termini of all influenza vRNAs (Uni13) and the 12-conserved nucleotides found at the 3' termini (Uni12) are shown in bold type. To generate the reverse-genetics plasmid, a 22 bp fragment containing 9 bp that correspond to the 5' termini of all vRNAs, followed by 4 bp (shown in gray) to create the Pst1 site, followed by 9 bp that correspond to the 3' termini of all vRNAs, was inserted between an RNA polymerase I promoter (pPol I) and terminator (Pol I-T). The RNA poly-merase II promoter and poly A signal, which flank the Pol I promoter and terminator in an opposing orientation, in this plasmid are not shown. The plasmid is digested with Pst1 and purified. The M-RTPCR amplicons and Pst1 linearized plasmid are mixed and treated with In-Fusion enzyme, and exonuclease activity exposes the complementary nucleotides at the termini leading to annealing and recombination (underlined nucleotides are the primer sequences used in M-RTPCR). The clones generated produce mRNAs from the RNA polymerase II promoter and authentic vRNAs are transcribed by RNA polymerase I upon transfection into appropriate host cells.

-GGCCCAATCATCATCTTTG

Fig. 2. Recombination-based cloning of M-RTPCR amplicons into reverse-genetics plasmids. Schematic diagram of the procedure used to clone M-RTPCR amplicons into our recombination-based reverse-genetics plasmid pBZ61A15. The M-RTPCR amplicons (1 segment shown) contain a dsDNA copy of a vRNA segment flanked by ten (5') or six (3') bp that are derived from the 5'-tails incorporated into the M-RTPCR primers (Uni13/Inf1 and Uni12/Inf1). The 13-conserved nucleotides at the 5' termini of all influenza vRNAs (Uni13) and the 12-conserved nucleotides found at the 3' termini (Uni12) are shown in bold type. To generate the reverse-genetics plasmid, a 22 bp fragment containing 9 bp that correspond to the 5' termini of all vRNAs, followed by 4 bp (shown in gray) to create the Pst1 site, followed by 9 bp that correspond to the 3' termini of all vRNAs, was inserted between an RNA polymerase I promoter (pPol I) and terminator (Pol I-T). The RNA poly-merase II promoter and poly A signal, which flank the Pol I promoter and terminator in an opposing orientation, in this plasmid are not shown. The plasmid is digested with Pst1 and purified. The M-RTPCR amplicons and Pst1 linearized plasmid are mixed and treated with In-Fusion enzyme, and exonuclease activity exposes the complementary nucleotides at the termini leading to annealing and recombination (underlined nucleotides are the primer sequences used in M-RTPCR). The clones generated produce mRNAs from the RNA polymerase II promoter and authentic vRNAs are transcribed by RNA polymerase I upon transfection into appropriate host cells.

tailed at the 5' terminus (6-9 nt), can be cloned into these pBZ plasmids (or any properly designed plasmid) using the procedures outlined below.

3.5.2. Preparation of Linearized Reverse-Genetics Plasmids

1. Prepare pBZ61A15 and pBZ61A18 plasmids with any commercial kits (e.g., QIAprep Spin Miniprep Kit) or standard alkaline lysis methods.

2. Digest 5 mg of each plasmid with 200 U (10 ml) of restriction endonuclease PstI, in a 300 ml reaction volume, at 37°C for 6 h.

3. Analyze 10 ml of the digestion by agarose gel electrophoresis to ensure complete digestion.

4. Purify the digested products using the QIAquick PCR purification kit following the procedure described in Subheading 3.3, but elute with 50 ml buffer EB instead of 30 ml.

5. Measure DNA concentration using spectrophotometer and dilute DNA to 50 ng/ml, aliquot multiple vials each containing 20 ml diluted DNA.

Typically ~4 mg of linearized plasmid DNA will be recovered from 5 mg of starting material.

3.5.3. Cloning of Amplicons into pBZ61A15 and pBZ61A18 or Other Similarly Modified Reverse-Genetics Plasmids (See Note 16)

1. Amplify the influenza A virus genome by M-RTPCR using the Uni/Inf primer set, as described in Subheading 3.2, using 28-30 temperature cycles total (see Note 17) and purify as described in Subheading 3.3.

2. Mix 200 ng of purified M-RTPCR amplicons with 100 ng of linearized pBZ61A15 and pBZ61A18 in two separate 200 ml PCR reaction tubes. Bring the volume to 7 ml each with ddH2O (see Note 18).

3. Add 2 ml of 5x In-Fusion reaction buffer and 1 ml of In-Fusion enzyme into each tube and pipette to mix completely but gently.

4. Use a temperature cycler to incubate the mixture at 37°C for 15 min and 50°C for 15 min, followed by 4°C hold.

5. Dilute the mixture with 40 ml TE buffer (pH 8), pipette to mix, and leave on ice.

6. Thaw competent cells on wet ice, add 3 ml of each diluted mixture into a corresponding vial of cells. Tap the vials gently to mix. Keep the vials on wet ice.

7. Incubate on ice for 30 min. Tap the vials every 10 min.

8. Incubate vials at 42°C (water bath preferred) for exactly 30 s, followed by immediate incubation on wet ice for 2 min.

9. Add 450 ml of room temperature S.O.C. into each vial.

10. Incubate the transformation mixture for 1 h at 37°C (shaking at 250 rpm is desirable).

11. Spread 20 and 200 ml of each transformation onto two labeled LB Agar plates containing 100 mg/ml of ampicillin (or appropriate antibiotic).

12. Incubate the plates inverted at 37°C for 16-20 h. Typically there are thousands of colonies on the 200 ml plate and hundreds of colonies on the 20 ml plate.

13. Screen for clones using standard PCR amplification across the insert region, or enzymatic digestion to identify the colonies containing the correct clones. If the nucleotide sequence is not determined for the 4th position of the 3' terminus of vRNA, then PB2, PB1, and PA clones should be selected from the pBZ61A18 vector, whereas HA, NP, NA, M, and NS should be selected from the pBZ61A15 vector.

3.6. TOPO TA Cloning Although the In-Fusion and other ligase-independent cloning Influenza Virus systems are amenable to any plasmid vector (including other

Amplicons reverse-genetics plasmids), there are a number of situations in which it is desirable to clone fragments into commercially available plasmids developed for specific functions (e.g., sequencing, RNA transcription, or protein expression). The TOPO TA Cloning Systems available (Invitrogen) are fast, efficient, and reliable for cloning any RT-PCR products, including M-RTPCR amplicons, into a wide range of plasmids (see Note 19). This system takes advantage of the fact that Taq DNA polymerases (including the high-fidelity version we recommend for M-RTPCR) add single non-templated nucleotides to the 3' termini of amplicons [often these are 3' adenosine (A) overhangs]. The vectors are provided as linearized plasmids with 3 ' deoxythymidine (T) overhangs that is bound to topoisomerase I, which efficiently ligates the inserts and plasmid. The 3 ' A overhangs of the PCR product complement the 3' T overhangs of the vector and allow for fast ligation (5 min).

3.6.1. TOPO TA Cloning 1. Amplify the influenza A virus genome by M-RTPCR using

Procedure either primer set as described in Subheading 3.2, using 28-30

temperature cycles total. Alternatively, amplify a specific segment using virus-specific terminal primers or segment-specific universal primers described by Hoffmann et al. (5) (see Note 20).

2. Concentration and purification of amplicons (optional, see Note 21).

(a) Prepare buffers for Zymo Research, Inc. DNA Clean and Concentrator™-5 as directed by manufacturer.

(b) Add 40-50 ml of M-RTPCR, or other RTPCR reaction, to 1.5-ml tube.

(c) Add 250 ml of the DNA Binding Buffer and mix briefly by vortexing.

(d) Transfer mixture to the Zymo-Spin™ Column in a collection tube.

(e) Centrifuge at 12,000-16,000 xg for 30 s. Discard the flow-through.

(f) Add 200 ml Wash Buffer to the column, and centrifuge at 12,000-16,000 x g for 30 s.

(g) Repeat wash step.

(h) Add 10 ml of ddH2O directly to the column matrix.

(i) Transfer the column to a 1.5-ml microcentrifuge tube and centrifuge at 12,000-16,000 x g for 30 s to elute the DNA.

3. Add 4 ml of amplicons from step 1, or purified amplicons from step 2, to a 0.2-ml PCR tube or a 1.5-ml tube.

4. Add 1 ml of salt solution (provided in kit).

5. Add 1 ml of the TOPO plasmid, mix by pipetting, and incubate 5 min at 22-25°C, then place on ice or store at -20°C.

6. Transform chemically competent Top10 E. coli (supplied) with 2-4 ml of the TOPO reaction as described in Subheading 3.5.3, steps 6-13.

4. Notes

1. Two (MBTuni-12 and MBTuni-12.4, or Uni12/Inf-1 and Uni12/Inf-3) of the three primers used in each set are nearly identical except for a single nucleotide (italicized). The A or G is used because the 3' termini of the vRNA segments vary at the 4th position (U or C), and this is dependent on the segment and the virus lineage. Often the polymerase segments have a C at this position and most other segments have a U, however this has not been extensively studied. It is recommended that the two primers be synthesized independently rather than synthesizing a primer with an R at this position (e.g., 5'-ACGCGTGATCAGCRAAAGCAGG-3'), so that the ratio of the two primers can be controlled.

2. Universal influenza A virus primers with 5' -tails that contain Mlu1 and Bcl1 restriction enzyme sites (Mlu1 is very rare in influenza A virus). The MBTuni primer set generally yields more robust amplification of all segments, but the ratio of small segment to large segments is greater than the Uni/Inf set. This primer pair is recommended for sequencing of the amplicons and can also be used for cloning of full-length segments into standard plasmids via ligation, TOPO TA, or ligase-independent cloning approaches.

3. The Uni/Inf primer set was designed primarily for cloning influenza A virus gene segments into reverse-genetics plasmids using ligase-independent cloning procedures, but can also be used for other downstream cloning and sequencing approaches. This primer set typically yields more uniform amplification of each gene segment.

4. If starting with cloacal swabs, we recommend isolation of virus by inoculation of eggs or TRIzol (Invitrogen) extraction with 10 mg of carrier tRNA.

6. Unequal concentrations of the Uni12/Inf1 and Uni12/Inf3 are used to improve amplification of the larger vRNAs (PB1, PB2, and PA). If using the MBTuni primer set, the same volumes should be used for MBTuni-12 and MBTuni-12.4.

7. The master mix was calculated for one additional reaction to account for small losses during pipetting.

8. The use of negative controls for both the RNA isolation process and the M-RTPCR reaction itself can help to identify the source of contamination if it occurs.

9. There are a number of variables that need to be considered and incorporated into the cycling parameters. The warm start reduces nonspecific amplification products. The regions of the oligos that hybridize with the influenza vRNAs are very short and A:T rich; therefore, 3-8 cycles with a lower annealing temperature (43-45°C) are required to incorporate the tailed primers into cDNA and for initiation of second strand synthesis. Finally the total number of cycles used varies depending upon the ultimate use of the amplicons. If the amplicons will be used for sequencing, 36 cycles (5 with an annealing temperature at 44°C and 31 with an anneal temperature of 57°C) are typically used. If the amplicons will be used for cloning purposes, 28 cycles (5 with an annealing temp at 44°C and 23 with an anneal temperature of 57°C) are typically used. Finally, the type of cloning procedure to be used should also be considered, if the products will be incorporated into TA-based cloning plasmids it is advisable to add a 10 min hold at 68°C between the 3rd stage and the hold at 4°C, because this will increase the percentage of the amplicons that have incorporated non-templated 3' adenosines.

10. We also find that the columns from Zymo Research, Inc. described later (Subheading 3.6.1) are very good for purification, particularly if you need to concentrate the amplicons.

11. The elution is very sensitive to pH. If using water to elute, make sure that the pH > 7. DEPC-treated water sometimes can have a quite acidic pH. Therefore, we recommend following QIAGEN's guidelines and use Tris-HCl pH 8.0 for elution of the DNA. We avoid TE because the EDTA can have inhibitory effects in some enzymatic reactions.

12. Some minor adjustments based upon the equipment and reagents used may be required, so you should discuss the sequencing of PCR products with your sequencing service provider.

13. Although M-RTPCR amplicons are well-suited for a high-throughput genomic sequencing using Sanger, or massively parallel technologies such as 454 sequencing, this section describes approaches for use by typical academic-type laboratories. Also, to sequence a complete genome one would need to setup ~10 M-RTPCR reactions. Another approach for complete genome sequencing is to use M-RTPCR for a first-round amplification and then use the amplicons as templates for overlapping PCR reactions with multiple primer sets. Primers for this type of approach were developed as part of the NIAID influenza genome sequencing project and can be found on the J. Craig Venter Institute website (http://gsc.jcvi.org/projects/ msc/influenza/infl_a_virus/primers.shtml).

14. A modified 12-plasmid reverse-genetics plasmid (pG26A12) derived from pHH21 was also produced and is described by Zhou et al. (7). We also refer to Chapter 12 for more details about the various reverse-genetics approaches.

15. The In-fusion enzyme has 3' -exo nuclease activity, so the 3'-overhang left from PstI digestion will be removed in the reaction and will not interfere with the recombination of the identical sequences between the amplicons and plasmids (Fig. 2). In a previous version, we used StuI (7) as the linearization enzyme; however, PstI is a more efficient enzyme than StuI.

16. Amplicons from segment-specific RTPCR reactions that have the same 5'-tails as the Uni/Inf primers can also be cloned into these pBZ plasmids. Additionally, virtually any plasmid (including most reverse-genetics plasmids) can be modified by the insertion of the In-Fusion cloning sites similar to those we have designed.

17. To reduce/limit mutations created during PCR, we use fewer temperature cycles for cloning procedures. Three-five cycles with an annealing temperature of 44°C are required to tail the DNA copies but the second stage can be reduced and this will reduce total yield but improve fidelity.

18. It is advisable to include the following controls in the In-Fusion reaction and transformation: (1) linearized vector only for In-Fusion reaction followed by transformation, (2) amplicon only for In-Fusion reaction followed by transformation, (3) pUC19 (included in the competent cell kit) control for transformation efficiency, and (4) mock-transformed bacteria as a control.

19. We recommend these systems primarily because they are straightforward and provide reproducible results.

20. The MBTuni primer set is recommended for this type of cloning because these primers contain restriction enzyme recognition sequences that are convenient for colony screening and subcloning into other plasmids.

21. Although concentration and purification of amplicons is not required for cloning RTPCR amplicons into TOPO TA plas-mids, we find that this can increase the number of positive colonies and thereby reduce screening efforts.

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