MYR 1030
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
`
`Page 1 of 30
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`i
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`liabil
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`Library of Congress Cataloging-in-Publication Data
`
`1. HIV in
`
`=
`
`-
`
`*
`
`+
`
`idee
`
`ruses) —
`
`(Viruses)
`‘seth
`“al
`fections
`2 HIV Infections
`1 “HIV. isolation earn :
`.
`4. HIV Seropositivity.
`virology. "3. HIV Infections — imam:
`5. Antiviral Agents.
`6. ae niques.
`QW168.5.H6
`H6761995
`616.97°9201—dc20
`
`QA201.A37H55 1995
`
`§5—15495
`
`ISBN 0 19 963493 9 (v. 1 : Hbk]
`ISHN 0 19 963492 O [v. 1 : Pbk)
`ISBN 0 19 963499 8 (v. 2: Hbk)
`ISBN 0 19 963498 X (v. 2 : Pbk)
`ISBN @ 19 963501 3 (set: Hbk)
`ISBN 0 19 963500 5 (set: Pbk)
`
`by Footnote Graphics, Warminster, Wilts
`Printed in Great Britain by Information Press Ltd, Eynaham, Oxon,
`
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`

`

`Sequence analysis of virus
`variability based on the poymerase
`chain reaction (PCR)
`
`ANDREW J. LEIGH BROWN and PETER SIMMONDS
`
`1. Introduction
`
`During most stages of HIV infection, the viral population contains a large
`number of closely related variants that have diverged within the indvidual
`over the course of HIV infection. For example, env sequences of human
`immunodeficiency virus in lymphocytes of infected individuals may show
`variability of up to 10% within the same sample (1, 2). Under these circum-
`stances, sequence analysis of PCR products amplified from such samples is
`often unreliable due to ambiguities in the sequencing gel. While there are
`methods for measuring the relative frequencies of alternative nucleotides at
`these polymorphic sites (see section 3), this does not distinguish the linkage
`relationships of the variant nucleotides so it is impossible reliably to recon-
`struct the constituent sequences.
`An even more serious problem is encountered when attempts are made to
`read a ‘consensus’ sequence in areas of the HIV genome where sequences
`differ from each by the presence ofinsertions or deletions of nucleotides(e.g.
`the V1, V2, V4, and V5 hypervariable regions of the env gene). The presence
`of variants that differ in length make a consensus sequence completely un-
`readable downstream of the position of the point of the deletion/insertion.
`On the other hand,
`the presence of length variants allow different viral
`populations to be rapidly compared, by amplification of a sample of the virus
`population across the hypervariable regions (3). Methods for analysing the
`length profiles of whole virus populations in vivo are described in section 3.
`
`2. Amplification of viral sequences
`2.1 Limiting dilution assays
`There are two methods for isolating single viral sequences to enable the
`analysis of heterogeneous samples. In the first method, cDNA orproviral
`DNAis amplified by the PCR followed by cloning of the PCR productinto
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`Andrew J. Leigh Brown and Peter Simmonds
`
`a plasmid (e.g. pUC) or bacteriophage (e.g. M13) vector. Sequencing can
`then be carried out on individual clones isolated by plating out the libraryin
`E. coli. Most of the problems encountered with this technique are associated
`with inserting blunt, non-phosphorylated DNA into a vector, and inaccuracies
`within the nucleotide sequences that are inevitably introduced by the amplifi-
`cation and cloning of viral sequences with certain DNA polymerases (sce
`section 2).
`These difficulties led us to develop an alternative method for obtaining
`single sequences from a heterogeneous sample, in which proviral DNA or
`cDNA present in a sample is diluted sufficiently prior to amplification to
`ensure that only single target sequences are amplified in each reaction. To
`separate the sequences,it is necessary to amplify DNA in multiple replicates
`at a dilution where only a small proportion of the reactions yield amplified
`DNA. Asthedistribution of very dilute DNA between tubes by macroscopic
`pipetting is a random stochastic process, the Poisson formula can be used to
`calculate the likelihood of positive tubes having originally contained one, or
`more than one molecules of target DNA (Table /). When the frequency of
`PCR-positive reactions is 0.2, approximately 95% of reactions can be pre-
`dicted to have originated from single target sequences, whereas only half
`of reactions will be derived from single sequences when the frequency of
`positives is 0.7.
`This method of separation is critically dependent on the reliable detection
`
`Table 1. Quantitation and separation of sequences by limiting dilution
`
`Observed
`frequency positives
`
`Calculated number
`DNA sequences”
`
`Proportion single
`copies”
`
`0.001
`0.01
`0.05
`0.10
`0.15
`0.20
`0.25
`0.30
`0.40
`0.50
`0.60
`0.70
`0.80
`0.90
`0.95
`
`0.001
`0.010
`0.051
`0.105
`0.163
`0.223
`0.288
`0.357
`0.511
`0.693
`0.916
`1.204
`1.609
`2.302
`2.996
`
`99.9%
`99.5%
`97.5%
`94.8%
`92.1%
`89.3%
`86.3%
`83.2%
`76.6%
`69.3%
`61.1%
`51.2%
`40.2%
`25.6%
`15.8%
`
`* Actual frequency, f (in target molecules/replicate tube) calculated according to
`the formula f = — In (f°), where (FP) is the observed frequency of negative
`reactions.
`® Proportion of positive replicates, f derived from a single copy of target DNA.
`
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`11; Sequence analysis of virus variability based on PCR
`
`of single molecules of the appropriate HTV sequence, While it is possible to
`detect positive reactions by hybridization of the PCR with a probe of high
`specific activity (4), the maximum sensitivity of such methods is generally
`around 3—100 copies of template DNA (5-7). We use the more sensitive
`nested PCR approach (Protocol /). A nested PCR can produce sufficient
`amounts of DNA from a single template molecule for sequence analysis. For
`example, a 300 bp target sequence has a molecular weight of 10000 g/mol
`and a mass of 0.016 ag (1 ag = 10~'* g); amplification by a factor of 10’ with
`outer primers, and 10° with inner primers would produce 160 ng of PCR
`product. In contrast, a single amplification of one copy of target DNA by as
`much as 10°-fold would produce barely enough product to be detectable by
`the most sensitive hybridization techniques (1.6 pg DNA).
`
`Protocol 1. Amplification of DNA by nested PCR
`
`
`
`Equipment and reagents
`« PCR buffer: 200 mM Tris-HCI pH 8.8, 500
`mM KCl, 15 mM MgCl,, 0.5% Triton X-100
`« Nucleotide triphosphates (100 = stock): 3
`dTTe
`mM each of dATP, dCTP. dGTP,
`15—60 pM sense primer (for a 20-mer, 100—
`400 pg/ml)
`15—60 pM antisense primer
`Mineral oi] (BDH, Cat. No. 279436)
`Taq polymerase (Cetus, Promega, North-
`umbria, Boehringer)
`Programmable thermal cycler
`Protective goggles, or (preferably) full face
`mask
`
`ned
`1.5% oegarose” gel in 1 = TBE,
`with 0.66 pg/ml ethidium bromide (note:
`ethidium bromide is highly mutagenic,
`always wear gloves when handling gels or
`solutions)
`1 = TBE electrophoresis buffer, containing
`0.66 pg/ml ethidium bromide
`size of
`Size markers spanning
`second PCR product (e.g. plasmid DNA,
`such as pBA322 digested with Haelll,
`Boehringer Cat. No. 821705)
`« Ultraviolet light box
`
`Method
`
`1. For each sample, pre-mix:
`e 5 pl PCR buffer
`38 yl water
`0.5 wl nucleotide triphosphates
`0.25 wl sense primer (approx. 4—16 pmol)
`0.25 pl antisense primer
`1 U Taq polymerase (0.1—0.25 pl at most manufacturers’ supplied
`concentrations)
`e 5 »!l DNA or cDNA sample (diluted, if appropriate)
`For amplifying multiple samples,it is easier to make up a stock contain-
`ing all but the sample, and adding 45 I to each tube. Cover with a drop
`of mineral oil.
`
`2. Transfer immediately to a thermal cycler set with the following temper-
`atures and times: 94°C 35 sec; 50°C 40 sec; 68°C 150 sec.
`
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`Andrew J. Leigh Brown and Peter Simmonds
`
`Protocol 1. Continued
`
`3. Samples should be amplified over 25—35 cycles (25 cycles is sufficient
`with outer and inner primers in nested PCR protocols).
`
`4. The samples should be heated at 68—72°C for 5 min at the end of the
`last cycle to terminate uncompleted strands.
`
`5. Amplified DNA is stable, and can be stored at 4°C (or at —20°C long-
`term) before carrying out the second amplification reaction or cloning.
`
`6. For each reaction, prepare 20 ul reaction buffer containing:
`2 p! PCR buffer
`17.7 wl water
`0.1 wl! of a 0.01 pg/ml orange G dye solution in water
`0.2 yl nucleotide triphosphates
`0.1 yw! inner sense primer (1.5—6 pmol, biotinylated)
`0.1 pl inner antisense primer (20 pmol, not biotinylated)
`0.4 U Taq polymerase (0.05—0.2 1)
`1 wl” of amplified DNA from step 5
`Cover with a drop of mineraloil.
`
`7. Transfer to a thermal cycler set with the times, temperatures, and
`numbers of cycles as for first PCR (steps 1—5).
`
`8. After amplification, analyse entire sample by agarose gel electro-
`phoresis, along with size markers. View on ultraviolet box. Bands
`ranging in size from 500—100 bp will be resolved adequately by this
`procedure.
`
`of the PCR.
`
`*High concentrations of agarose impair the transparency of the gel. Low melting point
`agarose produces clearer gels, but is more expensive, and not necessary unless gels are to be
`photographed.
`“Most laboratory workers experience the urge to add more than 1 pl to the second PCR
`reaction. In fact, this amount is more than adequate; addition of larger amounts will load to
`the appearance of multiple non-specific bands in the product, which can reduce the sensitivity
`
`To demonstrate the detection of single molecules of target DNA, serial
`tenfold dilutions of a cloned HIV sequence (pBH10.R3) were amplified with
`conserved gag-specific primers as described in Protocol | (Figures 1A and
`1B). While 65 fg of target DNA yielded a visible band after the first round
`of amplification (lane 3), as little as 6.5 ag (10~'* g) produced a positive signal
`after the second round (lane 7). The negative control that contained 1 pg
`herring sperm DNA was completely negative after the second round of
`amplification (lane 9). The mass of one molecule of the target DNA used in
`this titration (pBH10.R3) was 13 ag, thus the amount detected at the final
`dilution may have been as little as one molecule. We can also show that
`dilution separates single molecules of target sequence by amplifying replicate
`
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`11: Sequence analysis of virus variability based on PCR
`
`]
`
`2
`
`3
`
`4
`
`5
`
`6
`
`/
`
`8
`
`9
`
`10
`
`1
`
`$2
`
`3S
`
`ee Bee eee. BO)
`
`Se
`
`ae
`
`
`
`Figure 1. Titration of viral DNA sequences by nested PCR (reproduced with permission
`from the American Society for Microbiology). Dilution series of recombinant H!IV-1 DNA
`amplified by outer gag primers (A), followed by amplification of 1 wl! of product with inner
`gag primers (B). Lanes 1—8: tenfold dilutions of pBH10.R3 in 200 pg/ml herring sperm
`DNA from 6.5 pg in lane 1 (5 =x 10° copies target sequence) to 0.65 ag in lane 8 (nominally
`0.05 copies); lane 9: negative contro! (herring sperm DNA; lane 10: size markers (pTZ18R,
`digested with Haelll). Note:
`1 ag = 10~“* g. Addition of 65 fg (5000 copies) produces a
`faint band after the first PCR (lane 3), while 6.5 ag (nominally 0.5 copies) produces a band
`after the second PCR (lane 7). C. Limiting dilution: amplification of replicate samples each
`nominally containing 0.5 molecules of target HIV-1 sequence with nested gag primers (6.5
`ag amounts of pBH10.A3; lanes 2—12). Lane 1: negative control (1 wg herring sperm DONA);
`lane 13: positive contro! (65 ag pBH10.R3); lane 14: molecular size markers. See Tab/e 1
`for frequencies of positives at other input HIV DNA concentrations.
`
`samples containing different nominal amounts of target sequence, and analysing
`the products by agarose gel electrophoresis (Figure /C, and ref. 8). Multiple
`replicates of 6.5 ag amounts of target DNA yielded somepositivesignals and
`some negatives. Their frequency varied with the amount of target DNA
`amplified (8).
`
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`Andrew J. Leigh Brown and Peter Simmonds
`
`These experiments were carried out using recombinant DNA astarget.
`However, the method can be readily used for separation of viral sequences
`present in vivo. In order to ensure that the target viral sequences are dis-
`tributed randomly on dilution, it is usually necessary to shear DNA samples
`by ultrasonication or cleavage with a restriction enzyme that cleaves at rare
`sites (e.g. Sfil, Notl, that do not cleave HIV DNA). Separation of viral RNA
`sequences can be achieved by limiting dilution of the cDNA synthesized from
`it, as it is less likely to degrade on storage than RNA (9, 10).
`
`2.2 Solid phase purification of PCR product
`Sequencing using the Sanger method requires strand extension from a primer;
`this can be one of the primers used previously in the PCR, or it can corre-
`spond to sequenceswithin the amplified fragment. The sequencing reactionis
`highly sensitive to nucleotide triphosphates, primers, and buffer components
`present in the PCR product, thusit is necessary to purify the amplified DNA
`prior to carrying out the strand extension and termination reactions (11).
`Wehave adopted a single-strand purification procedure in which the PCR
`is carried out with a biotinylated primer in the second reaction (Protocol 2).
`Purification of the template in this way has a number of advantages.
`In
`particular, as there is a virtual absence of strong stops, strand extension takes
`place more readily and thus more of the sequence is readable. Less product
`DNAfrom the PCR is required to produce a strong signal in the sequencing
`gel, and the extraction of low molecular weight DNA attached to a solid
`phase is quicker and moreefficient than those based upon bonding to glass
`milk or ethanol precipitation. Finally, there are no problems with primercarry
`over; unreacted biotinylated primer remains attachedto the solid phase during
`the sequencing reaction, and can not cause inappropriate strand extension.
`
`Protocol 2. Purification of single-stranded amplified DNA on
`magnetic beads
`
`« 06.15 M sodium hydroxide (NaOH) solution
`
`Equipment and reagents
`« Extraction buffer: 10 mM Tris—HC! pH 7.5,
`1 mM EDTA, 2.0 M NaCi
`« Dynabeads coated with streptavidin
`® TE buffer: 10 mM Tris—HCi pH 8.0,
`EDTA
`
`1 mM
`
`« Magnet (e.g. Dynal MPC-E (Product No.
`120.04) for 1.5 ml Eppendorf tubes; Dynal
`MPC-96 (Product No. 120.05) for microtitre
`plates)
`
`A. Manual sequencing
`1. Amplify viral sequence(s) by nested PCR and one biotinylated (inner)
`primer (Protoco/ 1). Use a 50 yp! volume of PCR buffer for the second
`PCR.
`2. Resuspend dynabeads gently. For each sample to be sequenced, pipette
`10 wl into a 1.5 ml Eppendorf tube or into a well of a microtitre plate.
`
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`11: Sequence analysis of virus variability based on PCR
`
`Pre-wash in 40 ul 0.1% bovine serum albumin (caution: do not spin
`dynabeads during washing).
`3. Resuspend beads in 40 y! extraction buffer. Add 40 yl PCR product.
`Mix well, incubate at room temperature for 20 min, resuspending oc-
`casionally.
`4. Wash beads in 40 yw! extraction buffer; resuspend in 8 yp! 0.15 M NaOH.
`incubate at room temperature for 10 min.
`5. Remove supernatant (this contains unbound complementary sense
`DNA, and can also be sequenced (step 8)).
`6. Wash beads once with 50 »! NaOH, once with 50 yl extraction buffer,
`and once with 50 y! TE buffer.
`7. Resuspend beads in 20 yl TE buffer. Proceed to step 9.
`8.
`If required, neutralize unbound (complementary sense) DNA eluted
`from dynabeads by addition of 4 p! 0.3 M HC! and 1 pl 1M Tris—HCI
`pH 7.5 Make up to 20 pl with water.
`
`. Automatic sequencing (ABI 373A)
`
`. As above.
`Resuspend dynabeads gently. For each sample, pipette 20 pI! into a
`0.5 ml Eppendorf tube. Pre-wash in 40 yl extraction buffer.
`. Resuspend beadsin 40 yp! extraction buffer. Add 40—100 pl PCR prod-
`uct, according to concentration. Mix well, incubate at 48°C for 30 min.
`. Wash beadsin 40 pl extraction buffer; resuspend in 8 yl 0.1 M NaOH.
`Incubate at room temperature for 10 min.
`
`. As above.
`. As above but with 0.1 M NaOH.
`
`with water.
`
`. As above but in 14 yl sterile distilled water.
`. Neutralize complementary sense DNA eluted from dynabeads by addi-
`tion of 4 wl 0.2 M HCI and 1 pl 1 M Tris—HCI pH 7.5. Make up to 14 pl
`
`A potential problem with this technique is the limited binding capacity of
`the streptavidin bound to the solid phase, and it is essential that relatively
`low concentrations of biotinylated primers are used in the second PCR. We
`have found it important, especially for automated sequencing,to titrate the
`primers to find the minimum amountthat can be used reliably in the limiting
`dilution technique. For the V3 primers described in ref. 2, 6 pmol of sense
`and antisense primers have been used successfully in the second PCR for
`manual sequencing. For automated sequencing with T7 DNA polymerase,
`we use 5 pmol biotinylated and 10 pmol non-biotinylated primers.
`In most circumstances,it is desirable to sequence DNA from both ends of
`
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`Andrew J. Leigh Brown and Peter Simmonds
`
`the amplified fragment. This can be achieved either by carrying out two
`second PCR amplifications (with sense and antisense biotinylated primers) or
`by purifying both the bound and the unbound strand. These products are
`then separately extracted and sequenced from the opposite end with the other
`primer. To sequence the opposite strand the complementary single-stranded
`DNA is eluted from the solid phase after denaturation of the bound PCR
`product with sodium hydroxide (steps 5, 8). The eluate is neutralized by
`addition of an equimolar amount of HCI and Tris-HCl! buffer, adjusted to a
`final 20 wl volume, and sequenced in solution (steps 9 onwards).
`
`3. Sequencing PCR-amplified products
`3.1 Manual sequencing
`In the sequencing reaction for manual gels, the single-stranded DNA ts
`annealed to the sequencing primer; strand synthesis takes place initially in
`the presence of cold dGTP, dCTP, dTTP, and a limiting concentration of
`radiolabelled dATP, followed by a five minute incubation in the presence of
`each dideoxynucleotide for the termination reaction. The partial transcripts
`formed in each of the four termination reactions (i.e. with ddGTP, ddATP,
`ddTTP, and ddCTP) are separated by electrophoresis on a high-resolution
`denaturing (single-stranded) gel to allow the nucleotide sequence to be read.
`Single-stranded sequencing reactions are readable from around 10—20 bp
`downstream of the 3’ base of the sequencing primers, to around 350 bp using
`the BRL gel apparatus.
`
`3.2 Automated sequencing with dye-labelled
`terminators
`
`the approach
`Although also based on the Sanger sequencing procedure,
`involved in automated sequencing with dye terminators differs in some tm-
`portant respects. The four terminating dideoxynucleotides are labelled with
`a dye molecule (Protocols 3 and 4) each of which emits light at a different
`wavelength when excited by the laser which continually scans the gel while
`it is running. In consequence, the products of each reaction may be loaded
`in the same track, enabling 24 (or 36) reactions to be run on one gel. The
`scanning window, for excitation and detection of fluorescence is very small
`and each molecule being recorded only remains within the window for a very
`short time. Each reaction must havea similar intensity of signal. Consequently
`the yields from the template preparation steps must be carefully optimized
`for successful sequencing. The requirement
`is particularly important for
`sequencing with T7 DNA polymerase (‘Sequenase’). On the other hand, the
`gel products are continually scanned and a much longer read can be obtained
`as they are all being detected towards the bottom of the gel. Thus accurate
`sequencing is possible up to 450 bp and even longer fragments can be read if
`a lower level of accuracy can be tolerated. One major advantage of the dye-
`
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`11: Sequence analysis of virus variability based on PCR
`
`terminator method is the absence of ‘strong-stop’ artefacts, since any prema-
`turely terminated molecule will not have incorporated a labelled molecule,
`in contrast to manual Sanger sequencing.
`
`Protocol 3. Single-stranded DNA sequencing using the ABI 373A
`automatic sequencer and T7 DNA polymerase with
`dye-labelled terminators
`
`they are, to our
`
`knowledge, the only
`
`sup-
`
`Soph
`
`DNA template for six control reactions.
`
`Equipment and reagents
`* Materials for sequencing reactions are best
`« 8M ammonium acetate, filtered before use
`obtained from Applied Biosystems in the
`through a 0.2 um fitter
`PRISM Sequenase Terminator Single-
`« Formamide/EDTA
`(5:1):
`yaaaey DNA Semone ae At oe formamide/50 mM EDTA pH 8.0
`pliers of dye-labelled terminators for use
`* EYecermiinstorwash buffer: 0.01 M Tris pH
`with T7 DNA polymerase.
`The kits (enough for 150 reactions) comprise:
`—600 pl Sequenase dye terminator mix
`{a-thio dNTPs)
`—1.0 ml MOPS buffer pH 7.5: 0.4M MOPS
`pH 7.5, 0.6 M NaCl, 0.1 M MgCls, 25%
`glycerol
`—1,0 ml Mn?*/isocitrate solution: 50 mM
`MnCl,, 0.15 M isocitrate, 25% glycerol
`—160 pl Sequenase (1.35 U/l)
`together with primer and single-stranded
`
`deionized
`
`.
`
`:
`
`Method
`
`1. Prepare 5 = ss MOPS buffer by mixing equal volumes of the 0.4 M
`MOPS buffer and Mn?*/isocitrate solution (both supplied with the kit).
`This will slowly oxidize and should be prepared fresh daily.
`2. Mix the following in a microcentrifuge tube:
`e 5ul5 x MOPS
`* 1 wl Primer (0.8 ~M)
`e 14 yl template DNA
`Place in dri-block and heat to 65°C. Keep at this temperature for 2 min,
`then allow to cool slowly by switching off the dri-block. Centrifuge the
`tube to collect any condensation.
`3. Add 4 yl T7 dye terminator mix to the annealed template reactions and
`place in a 37°C heating block for at least 2 min to pre-warm the reaction
`ingredients.
`4. Add 1 «wl of the T7 DNA polymerase (1.5 U), keeping the tube in the
`block and mix well by pumping the pipette carefully several times.
`5. Cover tubes and continue incubation at 37°C for 10 min, then place on
`ice. Spin tubes to collect condensation.
`6. Remove excess dye terminator:
`(a) Biotinylated strand. Place tube in magnetic separator. Remove
`
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`Andrew J. Leigh Brown and Peter Simmonds
`
`Protocol 3. Continued
`supernatant. Wash DNAon beads two times with 50 p! dye termi-
`nator wash buffer. Wash once with 50 yp! TE and remove wash.
`Keep in the dark.
`(b) Non-biotinylated strand. Precipitate DNA by adding 29.5 pp! 8 M
`ammonium acetate and mix well. Add 150 1! 95% ethanol and place
`on ice in the dark. Spin in a microcentrifuge for 20 min. Remove
`supernatant and dry pellet by heating tube to 90°C for 2 min.
`Resuspend sample in formamide/EDTA by pumping with a pipette. For
`24 lane gel resuspend in 4 pl, for 36 lane gel resuspend in 3 pl. Store
`on ice until gel is ready to be loaded. Electrophorese on the sequencer
`(Protocol 4) using Filter set B.
`
`Protocol 4. Sequencing gel preparation for automatic sequencing
`
`Equipment and reagents
`
`ABI 373A automatic DNA sequencer: ABI
`glass plates, spacers, ge! casting comb,
`lower and upper buffer chambers, heat plate,
`alignment brace, 24- or 36-well sample
`comb, Macintosh computerwith at least 400
`Mb disk capacity
`Distilled or deionized water
`Electrical tape (1.5 inch)
`Urea
`40% acrylamide (frashly prepared)—can be
`bought as 30 g quantities from Bio-Rad
`{19:1
`acrylamide/bisacrylamide), which
`makes 75 mi 40% solution without the need
`for weighing out
`
`Binder clamps
`« Alconox™ (Aldridge)
`Mixed bed resin (Sigma)
`10 = TBE buffer: 0.89 M Tris base, 0.89 M
`boric acid, 0.02 M EDTA
`1 = TBE buffer
`Reusable bottle top filter unit (Nalgene)
`0.5 pm cellulose nitrate membrane filter
`(Whatman)
`Vacuum pump
`10% ammonium persulfate (APS)
`« TEMED
`
`50 mi syringe
`
`
`
`All reagents used should be ultrapure and high grade. Solutions should
`also be filtered to remove any particulate matter that might fluoresce or
`scatter light.
`
`Method
`1. Wash the glass plates, spacers, and comb with Alconox™ and warm
`water. The equipment should then be rinsed thoroughly with warm
`water followed by distilled water. Ethanol or other solvents should
`not be used as these contain fluorescent contaminants. Allow the
`plates to air dry.
`2. Align the plates with spacers between the side and bottom edges and
`then place a clamp on one of the long edges.
`3. Apply electrical tape tightly along the edge opposite the clamp wrap-
`
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`11: Sequence analysis of virus variability based on PCR
`
`ping the tape around both corners. Remove the clamp and then apply
`tape to the second long edge. Finally apply tape along the bottom
`edge. Any air pockets underthe tape should be eliminated and points
`of assembly likely to leak acrylamide solution should be reinforced.
`Particular care should be afforded to the bottom corners.
`
`. For a 60 mi gel solution (enough to pour one gel) mix the following
`reagents:
`e 30 9 urea
`« 9 ml 40% acrylamide stock solution
`e 20 mi distilled water
`e 0.5 g mixed bed resin
`
`. Stir for approximately 1 h until all the urea is dissolved.
`
`. Transfer the acrylamide solution to a 100 mi graduated cylinder con-
`taining 6 mI of filtered 10 = TBE buffer. Adjust the volume to 60 mi
`with distilled water.
`
`. Filter the acrylamide solution through a 0.2 .m cellulose acetate filter
`unit under vacuum, and then degas for 5 min. The degas time should
`be constantfor all gels, to ensure a reproducible polymerization rate
`for all runs.
`
`10.
`
`41.
`
`12.
`
`43.
`
`14.
`
`Gently pour the acrylamide solution into a 150 ml! beaker on ice.
`
`Add 300 »!l 10% ammonium persulfate (freshly made) and 33 ul
`TEMED, and gently swirl avoiding the generation of air bubbles.
`
`Immediately pour the solution between the plates using a 50 ml
`syringe and fill to about 3—5 cm from the top edge of the notched
`plate.
`
`Allow all air bubbles to rise to the surface and then place two clamps
`on either side of the plates.
`
`Insert the gel casting comb and then lay the plates in a horizontal
`position and secure the comb with three clamps. Place the clamps
`directly over the region of the gel casting comb that is sandwiched
`between the glass plates.
`Allow a minimum of 2 h to ensure complete polymerization prior to
`using the gel. The gel may be stored for a maximum of 24 h at room
`temperature although resolution will deteriorate if gels are stored any
`longer.
`Following polymerization remove the clamps, tape, and casting comb
`from the gel. Wash the plates thoroughly with tap-water to remove
`any excess acrylamide on the outside surface, and then rinse with
`distilled water. Ensure that all excess acrylamide is rinsed from the
`top edge of the gel and pay particular attention to the read region of
`the plates.
`
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`Andrew J. Leigh Brown and Peter Simmonds
`
`Protocol 4. Continued
`
`15.
`
`16.
`
`17.
`
`18.
`
`Allow the plates to air dry.
`Switch on the automatic DNA sequencer and open the data collection
`scan and map windows on the Macintosh computer. If the computer
`is networked it is advisable to reboot the computer before a sequence
`run is started.
`Place the lower buffer chamberinto the automatic sequencer and then
`insert the prepared sequencing gel and lock in place. Close thelid of
`the machine.
`Choose ‘Set Up Run’ on the automatic sequencer and verify the electro-
`phoresis conditions. Then choose ‘Configure’ and modify settingsas re-
`quired. Thefilter set for the sequence run can be modified from the
`‘Configure’ options. Filter set A for Taq sequencing and set B for T7
`sequencing.
`Scan the plates to check that they are clean. This can be done using
`the ‘Plate Check’ option from the ‘Start Pre-Run’ menu. If the baseline
`is not flat then clean the plates with distilled water, dry, and re-scan
`until the baselineis flat.
`. Check the PMT setting from the scan window (Y-axis of the lowest
`(typically the blue) line). This should be within the range of 800-1000.
`If outside this range then the PMT maybe adjusted by adjusting the
`PMTvoltage from the ‘Configure’ options on the sequencing machine.
`Conclude the plate check using the ‘Main menu’ option, ‘Abort Run’.
`Place and centre the sharks-tooth comb on the gel surface.
`Assemble the electrophoresis equipment fully and fill the upper and
`lower buffer chambers with 1 x TBE. Rinse the wells with a syringe
`filled with 1 x TBE buffer.
`Load 4 p! formamide/EDTA(for a 24 lane gel) and 3 yl (for a 36 lane
`gel) into the outer lanes on the sequencing gel. Pre-electrophorese
`the gel for 5 min (‘Start Pre-Run’, ‘Pre-Run Gel’).
`Prior to loading, denature the samples by heating at 90°C for 2 min,
`transfer immediately on to ice, and then load on the sequencing gel.
`The samples should be loaded in two batches,first the odd sample
`numbers and then the even. Sample wells should be flushed prior to
`each load with 1 x TBE buffer. The first batch of samples should be
`run for 5 min (‘Start Run’ on the keypad). The run should then be
`interrupted (‘Intrpt Run’) and the second batch of samples loaded and
`ran (‘Resume Run’).
`. Fill out the sample sheet for the sequence run on the Macintosh and
`then commence data collection.
`
`
`19.
`
`24.
`
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`11; Sequence analysis of virus variability based on PCR
`
`Sequencing with Tag polymerase (*Taqg-cycle sequencing’) has been de-
`veloped for both manual and automated procedures (Protocol 5). It is the
`most tried and tested procedure with ABI machines and can cope with much
`greater variation in template DNA concentration. However, it is subject to
`greater variability of signal between the four terminating nucleotides. This is
`compensated for by amplification of the signal of the weaker terminators
`within the ABI software, based on the overall level of signal for each. This
`compensation is ‘invisible’ (and inaccessible) to the operator. However, it
`has recently become apparent
`that
`the current version of the software
`assumes an even nucleotide composition, i.e. 25% of each nucleotide in the
`sequence. This is quite satisfactory for many applications, e.g. sequencing of
`mammalian genes, but should the sequence depart significantly from 25%
`base composition then artefacts arise. In the case of HIV, with its very high
`A + T content this presents a particular problem. Specifically, the signal for
`the C terminator is boosted to an inappropriately high level. In consequence
`background C peaks can occasionally be boosted sufficiently to interfere with
`the ‘correct’ peak at a site