i.,
`
`3
`
`t
`
`i 1
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`Research Report
`
`Quantitation of Targets for PCR by Use of
`Limiting Dilution
`
`PJ. Sykes, S.H. Neoh,
`M.J. Brisco, E. Hughes,
`J. Condon and A.A. Morley
`Flinders Medical Center,
`South Australia
`
`We describe a general method to quan-
`titate the total number of initial
`targets
`present in a sample using limiting dilution,
`PCR and Poisson statistics. The DNA tar-
`
`get for the PCR was the rearranged immu-
`noglobulin heavy chain (1'ng gene derived
`from a leukemic clone that was quanti-
`tated against a background of excess rear-
`ranged IgH genes from normal
`lympho-
`cytes. The PCR was optimized to provide
`an all-or—none end point at very low DNA
`target numbers. PCR amplification of the
`N—ras gene was used as an internal control
`to quantitate the number ofpotentially am-
`plifiable genomes present in a sample and
`hence to measure the extent of DNA deg-
`radation. A two-stage PCR was necessary
`owing to competition between leukemic
`and non-leukemic templates. Study of eight
`leukemic samples showed that approxi-
`two potentially
`amplifiabie
`leukemic IgH targets could be detected in
`the presence of 160000 competing non-
`leukemic genomes.
`The method presented quantitates the
`total number of initial DNA targets pres-
`ent in a sample, unlike most other quanti-
`tation methods that quantitate PCR prod-
`ucts. It has wide application. because it is
`
`INTRODUCTION
`
`Quantitation of DNA or RNA by the
`PCR is a problem that is presently un—
`der active investigation by many work—
`ers. Nearly all methods reported to date
`have used co—amplification of reporter
`DNA in the same tube and some form
`
`of quantitation of the amplified mate-
`rial (3,4,11). It is assumed that the effi—
`ciency of amplification of the reporter
`DNA is the same as that of the target
`DNA, and calculation of the amount of
`target DNA initially present is based on
`the amount of reporter DNA added or
`originally present and on the ratio of
`the quantities of target and reporter
`DNA as determined in the amplified
`material by various methods.
`We have been using the rearranged
`irnmunoglobulin heavy chain (IgH)
`gene as target DNA in the PCR to
`study patients with acute lymphoblastic
`leukemia (ALL) in order to detect and
`quantitate
`a minor population of
`leukemic cells within a larger popula-
`tion of normal lymphoid and non-lym—
`phoid cells (1,2). In a particular patient,
`all leukemic cells will have the same
`
`rearranged IgH gene that can act as a
`genetic marker to distinguish leukemic
`cells from normal non-lymphoid cells
`and T lymphocytes, which have not: re-
`arranged their IgH genes, and from
`normal B lymphocytes, which have un—
`dergone various and different rearrange-
`ments of their IgH genes. Quantitation
`of the unique rearrangement of the
`
`ond, gem-line IgI-I genes from cells,
`other than B lymphocytes, and rear-
`ranged IgH genes from normal B lym-
`phocytes will be present and, owing to
`homology with the leukemic sequence,
`may compete with it in the PCR. Our
`approach to quantitation has differed
`from that of other workers in avoiding
`use of reporter DNA and quantitation
`of amplified material. Rather, we have
`used the principle of limiting dilution,
`which is based on the use of a qualita—
`Live all—or—none end point and on the
`premise that one or more targets in the
`reaction mixture give rise to a positive
`end point. Accurate quantitation is
`achieved by performing multiple repli-
`cates at serial dilutions of the material
`
`to be assayed. At the limit of dilution,
`where some end points are positive and
`some are negative, the number of tar-
`gets present can be calculated from the
`proportion of negative end points by
`using Poisson statistics.
`In this paper we illustrate the use of
`limiting dilution analysis to quantitate
`a target for the PCR, using the rear-
`ranged IgH gene from a leukemic clone
`as an example. We also discuss four re—
`lated issues: optimization of the PCR to
`detect one or a few DNA targets; the
`effect of excess competing targets in
`the PCR; interference in the PCR by
`primers for an unrelated DNA segment;
`and the problem of DNA degradation
`in the sample. Although data from a
`small number of patients are included
`
`Ambry Exhibit 1011
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`
`MATERIALS AND METHODS
`
`DNA Samples
`
`PBLl DNA was obtained from nor-
`
`mal blood cells separated by Lympho-
`prepTM (Nycomed Pharma AS, Oslo,
`Norway) to contain predominantly nor-
`mal lymphocytes, and Ho DNA was
`from a bone marrow sample of a pa-
`tient with ALL obtained at diagnosis.
`Ho DNA provided a source of specific
`leukemic IgH targets and normal N—ras
`targets, and PBLl DNA provided a
`source of normal IgH and N—ras tar-
`gets. Ho DNA contained virtually
`100% leukemic cells, whereas PBLl
`was estimated to contain approxi-
`mately 15% normal B lymphocytes.
`The optimization experiments relied
`on DNA concentration of the Ho DNA
`
`sample (90 ng/pl) to estimate the num-
`ber of PCR targets present in the dilu-
`tions. Dueto the small amount of mate-
`rial available, an OD250 was not
`possible. The concentration of
`the
`DNA was obtained by ethidium bro—
`mide spotting against known DNA
`standards (Reference 8, Appendix E,
`p.6). Because one human diploid cell
`contains 6 pg of DNA (5),
`1 ug of
`PBLl DNA would contain approxi-
`
`mately 3.3 x 105 N—ras genes, approxi-
`mately 2.4 x 104 rearranged IgH genes
`and 3 x 105 unrearranged IgH genes.
`DNA from 7 other patients with
`ALL was extracted from flash bone
`
`marrow aspirate samples for patients 1,
`2, 3 and 7, from frozen Ficoll-Paque
`separated lymphocytes for patient 4
`and from stained, fixed bone marrow
`slides for patients 5 and 6. The DNA
`concentration of samples 1, 2, 3, 4 and
`7 was determined by 0D250 and for
`patients 5 and 6 by ethidium bromide
`spotting.
`
`PCR
`
`16.6 mM
`contained
`(7)
`PCRs
`(NH4)2SO4, 67 mM Tris—HCI, pH 8.8,
`10 mM fl-mercaptoethanol, 200 [Lg/ml
`gelatin, 2 mM MgClg, 0.1 mM each of
`deoxyadenosine triphosphate, deoxy-
`guanosine lriphosphate, deoxycytidine
`triphosphate
`and
`deoxythymidine
`triphosphate, 100 ng of each primer,
`varying amounts of template DNA
`(0.45 pg—l pg), and 0.4 U of Taq DNA
`Polymerase (AmpliTaq®: Perkin—El-
`mer, Norwalk, CT) in a volume of 25
`p1, overlaid with 25 pl light mineral oil.
`The samples were subjected to an in—
`itial 5 min denaturation at 94°C fol-
`
`lowed by varying numbers of cycles of
`1 min annealing at 55°C, 1 min exten-
`sion at 72°C and 1 min denaturation at
`94°C. A final 20 min extension at 72°C
`
`was performed at the end of each round
`of PCR.
`
`In all experiments, negative controls
`containing no template DNA were sub-
`jected to the same procedures to detect
`any possible contamination.
`
`PCR Primers
`
`Primers were synthesized on an
`Applied Biosystems Model 371 auto-
`mated synthesizer (Foster City, CA).
`Consensus primers (2) used to amplify
`all IgH genes in the first round of PCR
`were FR3A - 5’ ACACGGC(Cfl')(G./C)-
`TGTATTACI‘GT 3’ for the 3’ end of
`
`the V region; LIH - 5' TGAGGAGACG-
`GTGACC 3' for the 3' end of the I re-
`
`gion.
`Patient-specific primers (1) sited be-
`tween LJH and FR3A were used to am-
`
`plify Ho IgH genes only in the second
`round for the Ho DNA: Hol — 5-’ TGT—
`GCGAAAGAATCTC'I‘GCC 3’ for the
`
`3’ end of V; H02 — 5’ CCAGTAGTCA-
`AGGGTGGGTA 3’ for the 5’ end
`of J.
`
`Patient-specific IgH primers for the
`other seven patients will be published
`elsewhere.
`
`Specific primers were used for N-
`ms in both first and second rounds.
`
`- 5'
`First round: 231 (intron 1)
`AAGCTTI‘AAAGTACTGTAGAT 3';
`NB12 (exon 1) - 5’ CTCI‘ATGGTGG—
`GATCATA'ITCA 3’. Second round:
`
`NA12 (exon 1) - 5’ ATGACTGAGTA-
`CAAACTGGTGGTG 3’ that lies be—
`tween 231 and NB-l2 and in the sec—
`ond round is used with NB-12.
`
`For single-round PCR, N—ras was
`amplified by 231 and N312 and Ho
`IgH by H01 and H02. For two-round
`PCR, N-ras was amplified by 231 and
`NA12 followed by NB12 and NA12
`and leukemic IgH genes by FR3A and
`LII-I,
`followed by patient-specific
`primers in the second round for pa—
`tients 1—7. PCR products were electro-
`
`Number of first round PCR cycles
`NumberofmoleculesofPCRproductasseseeese'éaclMIdh"I0‘IDuC
`
`3 0
`
`4 0
`
`5 0
`
`10
`
`20
`
`Ambry Exhibit 1011
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`

`

`
`tgH(+PBLW
`
`Table 1. Sensitivity of Detection (Copies) of N—ms or IgH in the Presence or Absence of Competing
`Template
`
`Number of Copies Added"
`
`Number of Positives in 10 Tubes
`
`N-ms
`
`|gH(-PBL1)
`
`Quantitation
`
`Threefold dilutions of samples were
`prepared in water or PBLl DNA and
`10 replicates of each dilution were ana-
`lyzed using the optimal PCR protocol
`presented in this paper. The mean num-
`ber of targets required to give a PCR
`product was determined using the
`method of Taswell (10), which finds
`the Poisson distribution that best fits
`
`the data and provides an estimate (352
`test) of how well the data conform to
`that Poisson distribution.
`
`IgH
`
`37.50
`
`1 1.25
`
`3.75
`
`1.125
`
`0.375
`
`0.113
`
`N-ras
`
`75.00
`
`22.5
`
`7.5
`
`2.25
`
`0.75
`
`0.225
`
`Mean No. copies detected
`*Estimated from DNA concentrations
`
`RESULTS
`
`Preliminary Experiments
`
`Although one or a few N—ms or IgH
`targets could be detected by an opti-
`mized single-stage PCR, a mixing ex-
`periment showed that the addition of 1
`ug of PBLl DNA to provide excess
`competing non-leukemic templates de-
`creased detection of Ho templates to
`18% of that otherwise obtainable. A
`
`two-round PCR strategy using nested
`primers was therefore developed to im-
`prove sensitivity and specificity of am—
`plification.
`'
`
`Second-Round PCR -— Number of
`
`Cycles Required
`
`Serial dilutions were made to pro-
`duce aliquots that contained varying
`numbers of Ho IgH targets in 1 ug of
`PBLl DNA. Each aliquot was ampli-
`fied for 45 cycles in the first round, and
`104 to 10'9 dilutions of the product
`were made in water to produce a dilu-
`tion containing one or a few copies of
`amplified DNA as starting templates
`for a second round of 20—50 cycles. For
`IgH targets, amplified product could be
`detected from the 10'4 dilution after 20
`
`cycles, from the 10-5 dilution after 30
`cycles and from the 104 and 107 dilu-
`tions after 40 cycles. Amplified prod-
`uct could not be detected for the 10-8
`and 10'9 dilutions. These results indi-
`
`First-Round PCR — Number of
`Cycles Required and Effect of
`Competing Templates
`
`An initial experiment was per-
`formed using varying numbers of Ho
`IgH targets in 1 pg PBLl DNA, 20—50
`first-round PCR cycles, a 1:1000 dilu-
`tion between the first and second round
`PCR and 45 second—round PCR cycles.
`In this experiment, 30 first-round cy-
`cles were sufficient to enable an aver-
`
`age of 1.5 IgH targets to be detected.
`The quantitative aspects of the first
`round were then studied in more detail
`
`in order to determine both the optimal
`number of cycles and also the appropri—
`ate dilution to use between the first and
`second rounds of the PCR. Serial 10-
`fold dilutions of Ho DNA were made
`
`in water or in 1 ug of PBLl DNA. Be-
`tween 20 and 50 cycles of first-round
`PCR were performed, each aliquot of
`
`1234557391011
`
`amplified material was diluted 10'2 to
`10'12 in water and second-round PCR
`
`of 45 cycles was performed on each di-
`lution.
`
`The results are shown in Figure 1.
`Because the 45—cycle, second-round
`PCR gives detectable amplified DNA
`from one or a few targets, the maxi-
`mum dilution of the first—round mate-
`
`rial that still leads to amplification in
`the second round indicates the approxi—
`mate number of copies produced in the
`first-round amplification. A dilution of
`Ho DNA containing an average start-
`ing target number of 1.5 (IgH) and 3
`(N-ras) genomes,
`in the first-round
`PCR, resulted in a plateau of 109 copies
`after 40 cycles of amplification. How-
`ever, when other IgH targets were pre—
`sent, having been provided by dilution
`of starting material in PBLl DNA, am-
`plification of Ho DNA was less effi-
`cient and only 106 Ho IgH were pro-
`duced after 50 cycles of amplification.
`Based on these results, we decided
`to use 45 cycles for first-round amplifi-
`cation and a 10'3 dilution of amplified
`material between the first and second
`round. These conditions would be ex-
`
`pected to produce approximately 1000
`targets as starting material for the sec-
`ond round.
`
`Quantitation by Limiting Dilution
`Analysis and Poisson Statistics
`Serial dilutions of Ho DNA each in-
`
`volving 10 replicates were analyzed by
`
`Ambry Exhibit 1011
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`

`

`1.2
`
`1.6
`
`1.3
`
`86
`
`541
`
`23
`
`0.64
`
`0.12
`
`0.92
`
`2.28
`
`0.87
`
`0.41
`
`0.98
`
`7.67
`
`0.90
`
`0.35
`
`0.32
`
`0.57
`
`1.78
`
`0.37
`
`0.14
`
`0.87
`
`1 .05
`
`0.52
`
`
`
`Table 2. Sen-tivity of Detection (Copies) in Diagnostic ALL Patient Samples Using Two-Round
`
`Minimum Mean No. of Copies Detected
`
`Ratio N-raslllH
`
`Patient
`
`N-ras
`
`IgH (-PBL1)
`
`IgH (+PBL1)
`
`—PBL1
`
`+PBL1
`
`Geometric mean of ratios
`
`a1o replicates of dilutions studied.
`
`b5 replicates of dilutions studied for patients 1—7.
`
`cGeometric mean of all experiments in Ho are shown.
`
`are shown in Table 1. In two experi-
`ments, 1.7 and 2.6 copies of N-ras
`could be detected, and in three experi-
`ments, 3.5, 6.1 and 10 copies of Ho IgH
`could be detected in the presence of 1
`ug of PBLl DNA. The data in each ex-
`periment were consistent with a Poisson
`distribution (x2 test, p >0.05 for each).
`We studied sensitivity of detection
`in 7 additional diagnostic ALL patient
`samples. The results of all 8 samples
`are summarized in Table 2, and the in-
`tegrity of these DNA samples was in-
`vestigated by electrophoresis on a 1.3%
`agarose gel (Figure 3). In this table, the
`results in the columns referring to
`minimum mean number of copies de-
`tected are calculated from the total
`
`number of copies present as based on
`estimated DNA concentration.
`
`High sensitivity for detection of N-ras
`and IgH genes was observed in 5 pa-
`tients and lesser sensitivity in 3, pa-
`tients 5, 6 and 7. Because the number
`of N—ras copies detected depends on
`both the total number of copies which
`are present and the proportion which
`are amplifiable, the ratios of the num-
`ber of N—ras targets detected to the
`number of IgH targets detected give the
`proportion of potentially amplifiable
`
`competing targets, it proved possible to
`amplify a mean of 52%, i.e., to detect
`approximately two (11052) potentially
`amplifiable leukemic targets.
`Electrophoresis confirmed exten-
`sive DNA degradation in samples from
`patients 5 and 6 but not in patient 7, in
`whom the results, although somewhat
`variable, suggested a lesser degree of
`degradation. The DNA seen in Figure 3
`for patient 7, although faint, appears to
`be largely intact.
`
`Factors Affecting Amplification
`Efficiency
`
`Competing IgH targets. The effect
`of these in reducing amplification of
`the specific target has already been i1-
`lustrated (Figure 1 and Table 1).
`Competition between primer pairs.
`Several methods for quantitation of
`PCR targets rely on the use of two
`primer pairs in the same tube (3,4,11).
`To see whether this interferes with the
`
`efficiency of amplification and whether
`N-ras and IgH targets could be ampli-
`fied in the same tube, we quantitated
`low numbers of IgH targets in a two-
`round PCR by amplifying using IgH
`primers either alone or together with N-
`
`tectable (3.2 targets). However, in the
`presence of PBLl DNA, IgH primers
`alone were able to detect mean target
`numbers of 3.5 in one experiment and
`10 in a second, whereas with co-ampli-
`fication with N—ms primers, the IgH
`primers could only detect mean target
`numbers of 18 or 79. This effect of N-
`
`ras primers presumably resulted from
`the concurrent amplification of the 105-
`fold excess of N-ras targets provided
`by the PRU DNA.
`
`DISCUSSION
`
`general
`a
`have presented
`We
`method for quantitation of targets by
`PCR using the principle of limiting di-
`lution and use of Poisson statistics. For
`
`this approach, the PCR needs to be op-
`timized so that amplification will take
`place in an “all-or-none” fashion, and
`one or a few starting targets will give a
`positive result. When the optimal con-
`ditions are known, target concentration
`can be estimated by Poisson statistics
`applied to the results from replicate
`tubes taken at the limit of dilution.
`
`Limiting dilution analysis is widely
`used for quantitation in cell biology but
`is not commonly used for molecular
`quantitation. In 1990 Simmonds et a1.
`(9) reported its use to detect and quan-
`titate single HIV molecules. We were
`unaware of their report when we devel-
`oped the method presented herein, and
`review of the PCR literature suggests
`that the general approach is either not
`known or is misunderstood. Several
`
`other workers have performed serial di-
`lutions in their PCR studies and have
`
`used the term “limiting dilution.” How-
`ever.
`replicates have not been per-
`formed, the results have not been ana-
`lyzed
`by
`Poisson
`statistics
`and,
`importantly, the PCR has not been veri—
`fiably all-or-none because the reaction
`has been stopped after an arbitrary
`number of cycles, and there has been
`no knowledge of the number of targets
`that leads to either a positive or a nega-
`tive reaction. Single serial dilution with
`use of an arbitrary end point does not
`
`Ambry Exhibit 1011
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`

`

`Research Report
`
`targets against a background of highly
`homologous targets, e.g., detection of
`specific mutations in a population of
`normal cells. The biological problem in
`our study was the detection of rare
`leukemic cells in a large pepulation of
`nortnal cells, which in molecular terms
`became the problem of detection of a
`rare unique IgH sequence against a
`background of numerous other IgH se-
`quences. The two-stage PCR system
`that was developed proved capable of
`detecting approximately two (U052)
`potentially amplifiable leukemic IgH
`sequences against a background of ap-
`proximately 160 000 total genomes.
`These genomes would provide a vast
`excess of seqtrences that would com-
`pete with the leukemic IgH sequences
`for the PCR primers because they
`would contain approximately 2.4 x 104
`rearranged IgH genes from normal B
`lymphocytes and 3 x 105 germ—line
`IgH genes, each containing multiple V
`and J segments.
`Quantitation of PCR targets by lim—
`iting dilution can be compared with
`other methods for quantitation which
`use an added internal or external stan-
`
`dard, which is carried through the am-
`plification,- and which involve some
`form of quantitation of the amplified
`product(s)
`(3,4,11). Quantitation by
`limiting dilution does not require the
`use of an added standard, with the in-
`herent assumptions involved, and the
`end point
`is simple, nonq‘uantitative
`and nonradioactive. Furthermore, the
`end point is based on an all-or-none
`signal derived from the terminal pla-
`teau phase of the PCR, and the tech-
`nique is therefore relatively robust, be—
`ing able to cope with wide variations in
`amplification efficiency without affect-
`ing the estimation of DNA target num-
`ber. One potential disadvantage is the
`possibility of contamination, particu-
`larly if a two-stage PCR is performed.
`We use the precautions recommended
`by Kwok et al. (6) to minimize the risk
`of contamination. Replicate negative
`controls are used but also, in effect, di-
`lutions below the critical limit of dilu-
`
`tion act as additional negative controls.
`
`The assumption is that the extraction
`process does not modify the DNA.
`However, chemical modifications to
`DNA, such as strand breakage, deputi-
`nation or formation of adducts, may
`render the DNA incapable of acting as
`a template for the PCR. Quantitation by
`limiting dilution may have a unique ad-
`vantage in overcoming the potential
`problem of DNA modification because
`it is possible to also quantitate an endo-
`genous gene yielding a PCR product of
`similar size which is present in known
`number in all cells and which also un—
`
`dergoes the extraction procedure. In
`the present study the N—ras gene was
`selected as the endogenous gene to cor—
`rect for degradation. The 8 patients
`studied fell into 2 groups (Table 2). In
`5 patients, all or nearly all of the N—ras
`targets could be amplified, whereas in
`3 patients a les3er proportion of targets
`could be amplified. This suggested the
`presence of a variable degree of DNA
`degradation in these 3 patients, and this
`was confirmed in the 2 most obvious
`
`cases by electrophoresis (Figure 3).
`Nevertheless, as seen in Table 2, in all
`8 patients there was an approximately
`constant ratio between the number of
`
`ammifiable IgH targets and the number
`of amplifiable N-ras targets. These data
`suggest that the number of amplifiable
`N-ras genes, rather than the DNA con-
`centration, is the best indicator of the
`number of amplifiable genomes pre-
`sent, that virtually all potentially ampli-
`fiable leukemic IgH genes are ampli-
`fied in the absence of competing
`non-leukemic IgH genes and that ap—
`proximately half of the leukemic IgH
`genes are amplified in the presence of
`competing genes. Quantitation in the
`presence of DNA degradation can thus
`
`
`
`kbM1234567
`nerve-““h-
`2.3"
`
`
`
`0.6-
`
`be performed based on these data. Deg-
`radation can be a significant problem
`especially where fresh samples are un-
`available and to our knowledge this is
`the first reported PCR approach that
`enables correction to be made for it.
`Limiting dilution quantitation is
`simple, requires few manipulations of
`samples and has widespread applica—
`tion. Our usual approach is to perform
`a preliminary serial dilution experi-
`ment
`to determine the approximate
`point at which some amplifications are
`likely to be negative and some positive
`and then perform a detailed experi-
`ment, perhaps 40 tubes in all, involving
`multiple replicates of dilutions around
`this point. If quantitation is to be per-
`formed in terms of the number of am-
`
`plifiable targets of another gene such as
`N-ms, it should be noted that separate
`amplification reactions must be per—
`formed for each gene and that the vari—
`ance of the final value will be contrib-
`
`uted to additively by the variance of the
`estimation for each gene. More repli—
`cates will therefore be required for a
`given level of precision.
`
`ACKNOWLEDGMENT
`
`We thank Dr. R. Seshadri and Dr. I.
`
`Toogood for providing us with the pa-
`tient samples used in this study. This
`work was supported by the National
`Health and Medical Research Council
`and the Anti-Cancer Foundation of the
`Universities of South Australia. M.J.B.
`is in receipt of a Rotary Peter Nelson
`Leukemia Research Fund Fellowship.
`
`REFERENCES
`
`LEI-taco. MJ., 1. Condon, P.J. Sykes, S. Neck
`and A.A. Marley. 1991. Sensitive quantitation
`of neoplastic cells in remission and relapse
`marrows in B—lyi'nphoproliferative disease by
`use of the polymerase chain reaction. Br. J.
`Haematol. 79:211-217.
`
`2.Brisco. M.J.. L.W. Tan, AM. Orsborn and
`AA. Morley. 1990. Development of a highly
`sensitive assay. based on the polymerase chain
`reaction, for rate B-lymphocyte clones in a
`polyclonal population. Br.
`J. Haematol.
`
`Ambry Exhibit 1011
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`

`
`
`
`10.Taswell, C. 1931. Limiting dilution assays for
`
`
`the determination of immunocompetent cell
`THE 011”. Y DIFFERENCE
`
`
`frequencies. J. lmrnuno. 126:1614-1619.
`
`IS THE PRICE!
`
`11.Wang. A.M., M.V.L. Doyle and D.F. Mark.
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`1989. Quantitation of mRNA by the poly-
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`South Australia 5042
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`
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