WO2004053153A1 - Reactif et procede de determination de la taille de polynucleotides - Google Patents

Reactif et procede de determination de la taille de polynucleotides Download PDF

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WO2004053153A1
WO2004053153A1 PCT/AU2003/001640 AU0301640W WO2004053153A1 WO 2004053153 A1 WO2004053153 A1 WO 2004053153A1 AU 0301640 W AU0301640 W AU 0301640W WO 2004053153 A1 WO2004053153 A1 WO 2004053153A1
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polynucleotide
size
polynucleotides
dna
signal
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PCT/AU2003/001640
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English (en)
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Daniel Tillett
Carolina E. Beltran
Savant K. Karunaratne
Benjamin J. Briedis
Mathew Z. Damaere
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Nucleics Pty Ltd
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Priority claimed from AU2002953232A external-priority patent/AU2002953232A0/en
Priority claimed from AU2002953233A external-priority patent/AU2002953233A0/en
Priority claimed from AU2003901557A external-priority patent/AU2003901557A0/en
Application filed by Nucleics Pty Ltd filed Critical Nucleics Pty Ltd
Priority to AU2003285208A priority Critical patent/AU2003285208A1/en
Publication of WO2004053153A1 publication Critical patent/WO2004053153A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to a reagent and related method for detemiining the size of polynucleotides.
  • the invention relates to a polynucleotide of known size comprising one or more fluorescent moieties having a characteristic emission spectrum detectable in at least two data channels of a fluorescence-detecting instrument.
  • the polynucleotide allows for the data channels to be simultaneously utilised for detecting fluorescent emissions from the polynucleotide of known size and a sample polynucleotide.
  • a set of the polynucleotides forming a DNA ladder is particularly suitable for use in automated DNA sequencing applications since it allows for samples and size standards to be combined and analysed in the same data channels.
  • the related method of the invention allows for the dete ⁇ itiation of the size of a sample polynucleotide using the polynucleotide of the invention or a set of the polynucleotides of the invention with a fluorescence-detecting instrument, and is particularly suitable for DNA sequencing applications.
  • the standard approach to determine the size of an unknown DNA fragment is electrophoretic comparison with DNA fragments of known length.
  • Such sizing standards are generally prepared by restriction digestion of plasmid or bacteriophage DNA, or by polymerase chain reaction (PCR) amplification of well-characterized templates.
  • PCR polymerase chain reaction
  • STR Short Tandem Repeats
  • electrophoretic mobility of denatured DNA is primarily a function of fragment length, it is also influenced by several factors including the type of electrophoretic medium (solution vs. gel), type, concentration, polymerization and cross-linking of retarding molecules, buffers, electric field strength, and temperature (Bruland et al. 1999; Hahn et at 2001; Nataraj et al. 1999; Nock et al. 2001; Slater et al.2000).
  • the local Southern calibration formula or related formulae, are commonly employed to convert electrophoretic mobility to fragment length (Hepburn 1994;
  • Examples of systems that make use of labeled standards to size unknown DNA fragments include that of Caskey and Edwards (US patent 5,364,759).
  • This system uses labelled short tandem repeats of known size for sizing unknown DNA fragments.
  • Singer describes in US Patent 5,824,787 a reagent for generating a nucleic acid size marker that comprises repeats of subunits of defined regular intervals.
  • Schumm et al describe in US Patents 5,599,666, 5,674,686 and 6,156,512 a system for sizing short tandem repeats using an allelic ladder of known short tandem repeats.
  • Dau et al. describe in US Patent 6,013,444 a method of producing DNA standards derived from the locus to be measured which are co-electrophoresed with polymorphic DNA samples, either internally or externally, as bracketing, locus compatible, or specific DNA standards.
  • GENESCAN for use with the Applied Biosystem's slab gel 377 and capillary 3700 instruments, which uses fluorescent- labeled DNA size standards to estimate the sizes of unknown DNA fragments (Brondani and Grattapaglia 2001; Deforce et al. 1998; Mayrand et al. 1992; McEvoy et al. 1998; Mitchelson 2001). These instruments have the ability to detect the fluorescent emission from four different dyes in each lane, with each emission being recorded in separate data channels.
  • the GENESCAN system utilizes the instruments' capability for detecting multiple emissions in each lane to identify labelled standard in the same lane as unknown DNA fragments.
  • DNA sequencers can collect data in five channels (eg. the ABI 3700 and 3730 instruments). These sequencers allow the use of four data channels for samples (i.e. the channels that collect the A, G, C and T data in DNA sequencing applications) with the fifth channel being used for the sizing standard. While the use of five channel instruments allows more samples to be analyzed in parallel (le. four rather than three channels can be devoted to samples), current approaches require the use of a dedicated channel for the sizing standard and hence limit the overall efficiency of these systems.
  • Labeled size standards can be used to identify and correct mobility differences between unknown samples.
  • US Patent 6,110,683 describes the use of labeled size standards to correct non-linear mobility shifts in DNA sequencing reactions and improve base-calling accuracies.
  • Other examples include the use of labeled DNA ladders to size PCR products (Mayrand et al 1 92), HL single-stranded conformation analysis (Arguello and Madrigal 1999), CAG repeats (Annesi et al. 1997; Bruland et al. 1 99), and large DNA fragments under native conditions (McEvoy et al. 1998).
  • the size of a sample polynucleotide can be determined using the same data channels of a fluorescence-detecting iristrument to detect fluorescent emissions from both the sample and a size standard. This avoids the requirement to use a separate channel or lane for the size standard and provides clear advantages in terms of efficiency.
  • a fluorescent-labelled polynucleotide having a characteristic emission spectrum detectable in at least two data channels of a fluorescence-detecting instrument can be used as a size standard for this purpose and is disclosed herein.
  • a set of the polynucleotides of the invention eg. in the form of a DNA l dder of various polynucleotide sizes, is a particularly useful form of the invention and can be used, for instance, as a DNA sizing standard in applications where all data channels contain signal derived from labelled DNA fragments of unknown size, eg. in DNA sequencing reactions. Due to the characteristic emission spectrum of the polynucleotides of the invention, the location of the DNA ladder signal can be recognized even in the presence of signal from the sample allowing accurate base- calling. An algorithm for identifying the emission signals of the polynucleotides of the invention from those of the sample polynucleotides is disclosed herein and allows the size of the sample polynucleotides to be determined.
  • the invention allows for more efficient use of fluorescence-detecting mstruments as well as a reduction in reagents required resulting in significant overall time and cost benefits.
  • the invention allows for accurately base-calling DNA over a more extended size range than would ordinarily be possible and consequently for more DNA to be sequenced in a single run than with previously known technologies.
  • the present invention provides a polynucleotide of known size comprising one or more fluorescent moieties wherein the fluorescent moiety or moieties have a characteristic emission spectrum detectable in at least two data channels of a fluorescence-detecting instrument and wherein the characteristic emission spectrum is recognizable when one or more of the at least two data channels are utilised for detecting fluorescent emissions from a fluorescent-labelled sample polynucleotide.
  • the polynucleotide of known size may be of any composition but, preferably, it comprises deoxyribonucleotides. In another embodiment, the polynucleotide of known size comprises ribonucleotides.
  • the characteristic emission spectrum is detectable in three data channels of a fluorescence-detecting instrument. More preferably, the characteristic emission spectrum is detectable in four data channels of a fluorescence-detecting instrument. It may also, of course, be detectable in more than four data channels. Detection in different data channels is possible when the fluorescent moiety or moieties fluoresce at different wavelengths.
  • the characteristic emission spectrum is m the 400 to 800 nm wavelength range and, more preferably in the 500 to 700 nm wavelength range.
  • the 500-700 nm range is suitable.
  • the polynucleotide of known size comprises two fluorescent moieties. More preferably, the polynucleotide of known size comprises three fluorescent moieties.
  • fluorescent moieties useful in the present invention For example, many fluorophores suitable for labeling DNA are known in the art (e.g. those described in US patent 5,800,992; the contents of which are incorporated herein by reference). Clearly, the fluorophores used must be detectable in the equipment used for analysis. One of ordinary skill in the art will readily be able to identify fluorophores suitable for use, for example, with a particular DNA sequencer or other instrument. Ideally, the fluorescent moieties should possess a high fluorescence quantum yield for known absorption and emission maxima.
  • the fluorescent moiety or moieties selected cause nrinimal electrophoretic mobility changes to the polynucleotide to which they are attached.
  • the fluorescent moiety or moieties are selected from the following: carboxy-X-rhodamine, fluorescein, 6-tetramethylrhodamine-5(6)-caxbo ⁇ amide, BODIPY 493/503TM, BODIPY-Fl-XTM, (4 J 6-Dichlorotriazmyl)aminofluorescein, 6- carboxyfluorescein, 6-((5-dimemylannnonaphthalene-l-sulfonyl)artrino) hexanoate, Oregon Green 500TM, Oregon Green 488TM, Rhodol GreenTM, Oregon Green 514TM, Rhodamine Green-XTM, NBD-X, Tetrachlorofluorescein, Tetrabromosulfonefluorescein, BODIPY-F1 BR2TM,
  • the fluorescent moiety or moieties are capable of emitting an equal signal in all data channels of the fluorescence-detecting instrument. In practice, this may be difficult to achieve.
  • the characteristic emission spectrum comprises a relative contribution of 1.5, 2.8, 1.0, 3.6 in each of four data channels of the fluorescence-detecting instrument. In another embodiment, the characteristic emission spectrum comprises a relative contribution of 1,1, 2.0, 1.0, 2.5 in each of four data channels of the fluorescence-detecting instrument.
  • the four data channels are A, C, G and T data channels respectively of a DNA sequencer.
  • the polynucleotide of known size comprises the sequence: 5 ⁇ (ROX)(C6-NH)CTCCTTCTCTCCTT(dT-FAM)CTTTCTCCTCTTTC(dT-C6-NH- TAMRA)TCCTC-3' (SEQ ID No. 1).
  • the polynucleotide of known size comprises the sequence: 5'-(6-ROX)(C ⁇ -NH)CTCCTTCTCTCTT(dT-
  • the fluorescence-detecting instrument is a DNA or RNA sequencer, or a high performance liquid chromatography (HPLC), capillary electrophoresis (CE) or thin layer chromatography (TLC) machine. More preferably, the flu ⁇ rescence- detecting instrument is a DNA sequencer. Most preferably, the DNA sequencer is an ABI377, ABI3700 or ABI3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). When the DNA sequencer is an ABI 377, preferably the characteristic emission spectrum is detectable when filter set E is used.
  • the present invention provides a set of two or more polynucleotides of known size according to the first aspect
  • polynucleotides of the set of polynucleotides may be of any size, length or sequence and, for example, may be selected such that they form a "DNA standard" (also known as a "size standard” or "DNA size standard".
  • DNA standard also known as a "size standard” or "DNA size standard”.
  • One of ordinary skill in the field of the invention will be able to determine the ideal sizes, number and sequence of the polynucleotides in a set depending on the application.
  • the polynucleotides range in size from 5 to 10,000 nucleotides, nucleotide derivatives or nucleotide equivalent. In another embodiment, - li ⁇
  • the polynucleotides range in size from 10 to 5,000 nucleotides, nucleotide derivatives or nucleotide equivalents and in yet another embodiment, the polynucleotides range in size from 15 to 2,000 nucleotides, nucleotide derivatives or nucleotide equivalents.
  • the set of polynucleotides of known size comprises 2 to 500 polynucleotides. In another embodiment, the set comprises 5 to 200 polynucleotides and in a still further embodiment, the set comprises 20 to 50 polynucleotides.
  • the set includes polynucleotides of known size that are both larger and smaller than the predicted size of the fluorescent-labelled sample polynucleotide.
  • the set of polynucleotides comprises polynucleotides having a region of common sequence. More preferably, the region of common sequence comprises the fluorescent moiety or moieties. Most preferably, the region of common sequence is SEQ ID No. 1 or SEQ ID No. 5.
  • the present invention provides a method of determining the size of a fluorescent-labelled polynucleotide of unknown size using a fluorescence-detecting instrument, without the requirement to use a separate data channel for a size standard, comprising:
  • the present invention provides a method of determining the size of a fluorescent-labelled polynucleotide of unknown size using a fluorescence-detecting instrument, the method comprising:
  • the fluorescence-detecting instrument is a DNA sequencer.
  • the signal is derived in accordance with a predetermined algorithm.
  • the polynucleotides of unknown size are deoxyribonucleic acids.
  • a predetermined algorithm useful in the present invention may include the following steps:
  • step (a) is performed using Algorithm 1 as herein described.
  • steps (b) and (c) are performed using Algorithm 2 as herein described.
  • step (d) is performed using Algorithm 3 as herein described.
  • the method can be used for any suitable application.
  • the method is used for determining the size of polynucleotides generated for the purpose of DNA sequencing, RNA sequencing, genetic fragment analysis, linkage mapping, single-stranded conformational polymorphism (SSCP) , human identity testing, animal identity testing, DNA or RNA sizing or paternity testing. More preferably, the method is used for detemn ing the size of polynucleotides generated for the purpose of DNA sequencing.
  • SSCP single-stranded conformational polymorphism
  • the present invention provides a method of synthesizing a polynucleotide of known size according to the first aspect or a set of polynucleotides of known size according to the second aspect wherein the polynucleotide or polynucleotides are prepared by linking a first polynucleotide carrying the fluorescent moiety or moieties to a second polynucleotide of predete ⁇ nhied size.
  • the first polynucleotide is linked to the second polynucleotide via a t ⁇ d polynucleotide.
  • the first polynucleotide is a chemically synthesized polynucleotide.
  • Standard protocols for synthesizing polynucleotides are available and will be known to the skilled addressee including thephosphoramidite chemistry method (Gait 1991; Sproat 1995; Ve ⁇ na and Eckstein 1998).
  • the first polynucleotide has the sequence shown in SEQ ID No, 1 ,
  • the first polynucleotide has the sequence shown in SEQ ID No. 5.
  • a polynucleotide complimentary to the first polynucleotide is hybridized to the first polynucleotide.
  • the first polynucleotide has the sequence shown in SEQ ID No. 1
  • the polynucleotide complimentary to the firstpolynucleotide has the sequence shown in SEQ ID No. 2.
  • the polynucleotide complimentary to the first polynucleotide has the sequence shown in SEQ ID No. 6.
  • This double-stranded polynucleotide can be ligated to DNA fragments that have been generated from restriction enzyme digestions.
  • the present invention provides a polynucleotide of known size labelled with two or more structurally defined fluorescent moieties such that the fluorescent emission of the labelled polynucleotide occurs at a greater number of wavelengths than that emitted by a polynucleotide labelled with any one, structurally defined, fluorescent moiety.
  • the present invention provides a method of determining a signal from a series of data channels indicative of the location of a fluorescent-labelled polynucleotide of unknown size relative to a polynucleotide of known size, the polynucleotide of known size having one or more fluorescent moieties that are detectable in at least two data channels, the method comprising the steps of:
  • the present invention provides a method of determining the size of at least one unknown DNA fragment, the method comprising the steps of:
  • step (c) further comprises the steps of:
  • step (cl) further comprises the step of:
  • the extraction process includes:
  • the curve fitting includes accounting for the blurring of peaks in the rescaled signal.
  • the method is carried out simultaneously on separate series of unknown DNA fragments.
  • uriknown DNA fragment refers to polynucleotides of unknown size.
  • DNA ladder means a set of polynucleotides of known size.
  • size insofar as it relates to polynucleotides means the number of nucleotides, nucleotide derivatives or nucleotide equivalents present in the polynucleotide.
  • the term "set" insofar as it relates to polynucleotides means two or more polynucleotides.
  • nucleotide includes within its meaning molecules made up of two or more nucleotides, nucleotide derivatives and or nucleotide equivalents.
  • nucleotide derivative refers to any molecule derived from a naturally-occurring nucleotide and includes, but is not limited to: 2'-deoxy-5-pro ⁇ yluridine, 8-o ⁇ o-2'- deoxyguanosine, 5-hydoxy-2'-deoxy ⁇ uidine, or 6-(2-deoxyl-beta-D ⁇ ribofuranosyl)-3,4- d ya o-8H-pyi rnido-[4,5-c][l,2]oxazin-7-one.
  • nucleotide equivalent includes within its meaning a molecule similar, or identical to, a i turaUy-occurring nucleotide in terms of its structure and or conformation and or function and/or overall effect on behaviour of a polynucleotide in which it is present - behaviour of the polynucleotide being behaviour, for example, in terms of electrophoretic mobility.
  • nucleotide equivalent includes, but is not limited to: a nucleotide or nucleotide derivative to which a fluorescent moiety is attached, a peptide- nucleic acid (PNA), a locked nucleic acid (LNA), a ⁇ -O-methyl rNTP, a thiophosphate linkage, an addition to the amines of the bases (e.g. linkers to functional groups such as biotin), a non-standard base (eg. arr ⁇ ho-adenine, iso-guanine, iso-cytosine.
  • PNA peptide- nucleic acid
  • LNA locked nucleic acid
  • ⁇ -O-methyl rNTP a thiophosphate linkage
  • an addition to the amines of the bases e.g. linkers to functional groups such as biotin
  • a non-standard base eg. arr ⁇ ho-adenine, iso-guanine, iso-cytosine.
  • a chromatographic trace generated from 2 ng of the Sat ⁇ A/MspI MEFL DNA standard, showing the relative signal contribution of the MEFL DNA standards in the A, C, G and T channels.
  • Figure 2A shows a region of a chromatographic trace data resulting from the addition of 2 ng of the SauiMMspl MEFL DNA standard to a pGEM3Z(f+) DNA sequencing reaction,
  • the trace data has been baselined and crosstalk corrected using the supplied BigDye vers.1 matrix.
  • single peak derived from the MEFL DNA standard can be seen at approximately scan number 3500. This peak has been indicated with an arrow.
  • Figure 2B shows the same region after processing the trace data using the steps described in Algorithm 2.
  • a single peak can be observed at approximately scan number 3500 indicating the presence of a MEFL standard peak at this location. This demonstrates that a MEFL DNA standard can be identified and the peak modelled even in the presence of signal in all four er ⁇ ission channels.
  • Figure 2C shows the chromatographic data after application of Algorithms 1, 2, and 3, followed by mobility shift correction and inter-channel normalisation. At the prior location of the MEFL DNA standard peak, only one major peak is retained (in the G channel). This is indicated by an arrow.
  • Figure 3 A shows the region of the chromatographic trace from scan number 0 to 910 derived from an Sau3A AM MEFL DNA ladder mixed with the internal-lane size marker GENESCAN-ROX 500 (Applied Biosystems).
  • GENESCAN-ROX 500 has fragment molecular lengths of 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 nucleotides.
  • Two peak types were observed: 1. Peaks that fluoresced equally in the C and T channels (ROX). 2. Peaks that fluoresced in all four channels (MEFL DNA standard). The ROX derived peaks are marked with a * (star) and the MEFL standard derived peaks with arrows.
  • Figure 3B shows. the section of the trace coiresponding to scan numbers 0 to 910 when the chromatographic trace data of Figure 3A was processed using Algorithm 2 and the MEFL standard relative emission contributions calculated in Example 1.
  • Figure 3C shows the chromatograph trace data after further processed using Algorithm 3 and the MEFL DNA standard peak location calculated previously.
  • ROX derived peaks were observed at scan numbers 10, 191, 383, 690, 795, and 867 in this section of the trace file, corresponding to the 50, 75, 100, 139; 150 and 160 nucleotide ROX size markers.
  • FIG. 4 Use of the MELF DNA standard to size FCR amplified microsatellite fragments.
  • FIG 4A shows the region of a chromatographic trace from scan number 1480 to 3440 for PCR amplified inicrosatellite fragments mixed with GENESCAN-TAMRA 500 and the SauZAIMspl MEFL DNA ladder.
  • Three peak types were observed 1. Peaks that fluoresced equally in the C and A channels (TAMRA).2. Peaks that fluoresced in all four channels (MEFL DNA standard). 3. Peaks that fluoresced in the G channel (microsatellite).
  • the TAMRA derived peaks are marked with a * (star), the MEFL standard derived peaks with arrows, and the two microsatellite peaks with P 1 and 2.
  • Figure 4B shows the chromatograph trace data (scan numbers 1480 to 3440) which resulted when the chromatographic trace of Fig 4A was processed using
  • MEFL DNA standard peaks were observed at scan numbers 1054, 1307, 1328, 1342, 1541 and 3347 corresponding to the 182, 205, 207, 209, 227, 394 nucleotide sized fragments.
  • MELF derived peaks were observed at scan numbers 1541 and 3347 (indicated with arrows), corresponding to the 227 and 394 nucleotide MELF size standard markers.
  • Figure 4C shows the chromatographic trace that resulted when the MEFL DNA standard data (Fig 4B) were removed from chromatographic trace data shown in Fig 4A.
  • SEQ ID Nos. 1 to 6 are shown- The position of sequence CTGCGCTCGG in SEQ ID No, 3 (see Example 3) is underhned.
  • A A single peak obtained from the chromatographic trace generated using 51 finol of the OLIGO 1, showing the relative signal contribution of OLIGO 1 in the A, C, G and T channels.
  • B A single peak obtained from the chromatographic trace, generated from 100 fmol of the OLIGO 5, showing the relative signal contribution of OLIGO 5 in the A, C, G and T channels. It can be seen that the relative signal contributions from OLIGO 5 are in a narrower range than those from OLIGO 1. In particular, it can be seen that the signal for the G channel increases relative to the other channels when OLIGO 5 is used. The calculated signal contributions are 1.1(A),
  • Figure 7 Determination of the relative signal contributions of OLIGO 1 and OLIGO 5 across the four channels (A, C Pain G and T) when analysed on am ABI3730 DNA sequencer.
  • A A single peak obtained from the chromatographic trace, generated from 31 f ol of the OLIGO 1, showing the relative signal contribution of the OLIGO 1 in the A, C, G and T channels.
  • B A single peak obtained from the chromatographic trace, generated from 15 fmol of OLIGO 5, showing the relative signal contribution of the OLIGO 5 in the A, C, G and T channels. It can be seen that the relative signal contributions from OLIGO 5 are in a narrower range than those from OLIGO 1. In particular, it can be Seen that the signal for the G channel increases relative to the other channels when OLIGO 5 is used.
  • the calculated signal contributions are 1.0(A),
  • a reagent f r determining the size of unknown DNA fragments run on automated DNA sequencers or other instruments used for analysing DNA fragments without the need for a separate channel or lane is described.
  • the reagent is formulated from a synthetic oligonucleotide that has been labelled with three fluorophores.
  • the fluorophores are chosen such that their emission spectrum, when analysed on an automated DNA sequencer, is present in four data channels.
  • the multi- emission channel fluorogenic oligonucleotide (or "multi-emission fluorescent-labelled” (MEFL) polynucleotide) is joined to a series of DNA fragments of known size creating a fluorogenic "DNA ladder” , "DNA standard”, or multi-emission channel fluorogenic labelled DNA ladder (referred to as the "MEFL DNA ladder” or the “MEFL DNA standard”).
  • the MEFL DNA ladder can be included in the loading dye formulation, or at other appropriate stages of sample preparation, for each sample for every lane to be run on a DNA sequencer.
  • the signal derived from the MEFL DNA ladder fragments is identified, and hence disciirninated from, signal derived from the sample DNA fragments by virtue of its characteristic emission spectrum across the four data channels of the DNA sequencer.
  • the signal derived from the MEFL DNA standard fragments could prevent identification of the unknown DNA fragment, the signal is ⁇ dgorithmically removed via subtraction of signal proportional to the channel emission ratio of the MEFL DNA standard.
  • the standard loading dye used on automated DNA sequencers is mixed with the MEFL DNA ladder but the MEFL DNA ladder can be included at any stage in the preparation of the sample that is convenient.
  • the MEFL DNA ladder is constructed from a synthetic oligonucleotide that has been synthesized so as to contain three fluorophores of different emission spectra such that the emission spectrum obtained from the MEFL DNA ladder occurs simultaneously in four channels.
  • the labeled oligonucleotide is joined to a series of known-sized DNA fragments such that a MEFL DNA ladder of known size is generated.
  • the characteristic multi-channel emission spectra of the labeled DNA fragments provide a means of discriminating signal derived from the MEFL DNA ladder fragments from signal derived from the unknown-sized DNA sample fragments.
  • the characteristic multi-channel emission spectra provided by a particular MEFL DNA ladder on a given sequencer can be determined by electrophoresis of the MEFL DNA fragments on the sequencer. The location of the peaks derived from the multi-labeled fragments can be identified and the signal ratios determined at the locations of the peaks for each of the channels in which it emits fluorescent signal.
  • This process provides a multi-channel emission signature that is specific for the fluorescent dye combination and DNA sequencer used. While this signature is ideally equal in all measured channels, it is not required for generation of the emission signature. This process can be performed using Algorithm 1 :
  • the crosstalk matrix, K is determined by examination of the signal obtained in each channel when the individual dyes used are run on the automated DNA sequencing insttu ent alone. This process provides the degree of crosstalk in signal that occurs in each channel derived from each dye used in a particular experiment
  • the signal Qjt(x) can be considered a N-row by ⁇ f-column matrix on which operates.
  • this matrix is an identity matrix with no crosstalk between channels, but in reality the non-diagonal elements of the matrix are non-zero.
  • the standard signal Qj ⁇ (x) is operated on by inv(K) to obtain a new crosstalk corrected MEFL DNA standard signal. Equation 1
  • the location of signal derived from the MEFL DNA standard can be identified using Algorithm 2.
  • Electrophoreses of labelled DNA or RJNA fragments (either of unknown or known size) on an automated DNA sequencer in the presence of the MEFL DNA standard ladder generates a four channel chromatogram (trace) U_n(x) of M data points.
  • This trace is a simple superposition of the emission signal obtained from the labelled DNA or RNA fragments, qjt(x), with the emission signal obtained from the MEFL DNA standard, Q_n(x)' ⁇
  • T_n(x) Sj ⁇ (x) + s_n(x)
  • $_n(x) is the crosstalk removed form of the unknown signal without the MEFL DNA standard.
  • the aim of the algorithm is to obtain s_n(x), using T_n(x) and S_n(x).
  • Baselining the unsealed trace T t(x) generates B_n(x). This is performed by median filtering each channel, n, of the trace and then dividing into windowed sections. In each windowed section, the irnhima, as well as mid-points of valleys, are found. The intensity of the point with the second lowest intensity within the window is subtracted out as a constant from the windowed section of the channel to produce the base ⁇ ined trace, Bjt(x).
  • a blur function, b( ⁇ ), is constructed from Bjt(x) by fitting a quadratic curve via an implementation of the Marquardt-Levenberg non-linear least squares algorithm (Levenberg 1944; Marquardt 1963) to the set of points obtained by measuring the widths of peaks generated from the labelled DNA or RNA fragments at 50% of the peak height at each peak location. Peaks can be identified by selecting signal maxima in each channel that have intensities above the average channel intensity in that channel. Each resulting peak is then traversed to its left and right while the intensity of the signal monotonically decreases until the signal intensity equals half the peak height on either side of the peak.
  • the distance between the resulting left and right points is the full-width-at-half-maximum value of the peak (this hereby defined as the "Peak Width ").
  • Double peaks or multiple peaks in the trace, formed due to the merging of two or more adjacent peaks in a given channel, are eh inated by an outlier removal process.
  • the Peak Width values from all channels are combined and a quadratic function is fit to the Peak Width values.
  • Outlying Peak Width values are eliminated by comparison to the fit function and a new quadratic function fit is obtained.
  • the resulting function, b(x) is a measure of the degree of blur of a given peak at location x in the trace.
  • the contribution-scaled trace Tj ⁇ (x) is obtained by scaling each channel of the crosstalk corrected trace by the inverse of the respective standard contribution value.
  • the Minimum Channel, M(x), is obtained from the contribution-scaled trace
  • mji is the largest intensity value for each channel n for the sake of normalisation.
  • Mg(x) is obtained from M(x) by Gaussian shape filtering and correlation with a Gaussian function (Whittaker and Robinson 1967) using the functions described in Equations 6 and 7
  • Mg(x) -sum_fx' x-r to x+r ⁇ M(x') g(x',x) /sum_ ⁇ x' - x-r to x+r ⁇ M(x)'
  • Mh(x) is generated by cross-correiation filtering of Mg'(x) using a Caucby function (Papoulis 1984) described in Equations 9 and 10
  • a filtered standard channel Mh'(x) is generated by cross correlating Mg '(x) with two signals obtained by shifting Mh '(x) to the left and right by a distance d(x) where d(x) is a function obtained by constructing a straight line fit to the displacements that give the best correlation at windowed sections of the channel. Equation 11
  • the expected number of standard peaks, P, in the trace for a given trace length can be determined.
  • the location of the MEFL DNA standard fragments can be determined by identifying the P tallest peaks in Mh '(x).
  • the size of the unknown DNA sample fragments can b determined by comparison to the location of fragments of the known-sized MEFL DNA standard.
  • the local Southern calibration formula, or related formulae can then be used to convert the electrophoretic mobility of the unknown-sized sample fragments to a length in base pairs (Hepburn 1994; Man et al, 2000; Mayrand et al. 1992; Rosenblum et al. 1997; Syndercombe Court et al. 1992).
  • This formula uses the local segment of the calibration curve, as determined by the nearest neighboring multi-labeled marker fragments which bracket the unknown sample, to generate an accurate measurement of the unknown fragments.
  • the signal arising from the unknown DNA or RNA fragments at ih$ locations of the MEFL DNA standard peaks can be determined using Algorithm 3.
  • Step 1 The regions around each MEFL DNA standard peak location are selected by measuring the width of each peak at each of the specified locations at 20 percent of the maximum peak intensity. For each MEFL DNA standard peak, a set X is generated of those points xj in each channel that belong to the region of the MEFL DNA standard peak.
  • Step 2 T'_n(x) is obtained by scaling each channel by its predefined MEFL DNA standard contribution cji as defined by Equation 4.
  • Step 3 Using the MEFL DNA standard peak locations an approximation R(x), of the MEFL DNA standard signal Sjx(x), is constructed. This - ⁇ proximation is obtained from the lowest value, point by point, of the four, contribution-scaled, chromatogram channels T'j ⁇ (x) for each member x_i of the set X defined for each MEFL DNA standard peak. Any points not part of the set ⁇ Tare made equal to zero.
  • R( ⁇ ) is equal to the MEFL DNA standard signal in the channel where the standard has the weakest contribution, S_weakest(x).
  • Step 4 At each MEFL DNA standard peak location, a peak modelled using Equation 14 (the Cauchy distribution) is fit by an implementation of the Marquardt- Leveriberg nonlinear least squares algorithm to R(x) (Levenberg 1944; Marquardt 1963). The amplitude alpha of the Cauchy distribution is held fixed and equal to the intensity of R(xo) while the centre xo and width beta parameters are allowed to be free.
  • R'(xJ) (P(xJ) R(x_i) A 2) (l/3), x_i s X.
  • Step 5 The estimates of the minimal MEFL standard signal in the regions around each MEFL DNA standard peak location, R'( ⁇ ), are combined by superposition to produce the final determination of the MEFL standard signal for the channel with the weakest contribution, R_we ⁇ kest(x).
  • Step 6 R_weakest(x) is used as an appropriate estimate of S_weakest(x).
  • MEFL DNA standard signal is removed from each channel by scaling R_weakest(x) by its relative contribution c_n and subtracting from T_n(x), generating s ⁇ (x). Equation 16
  • s_n(x) is an approximation of the chromatographic trace obtained from the unknown DNA fragments in the absence of MEFL DNA standard. It can therefore be used in any application where data is to be obtained from the unknown DNA fragments (e.g, determining the order of nucleotide bases in a DNA sequence, or the absolute size of unknown DNA fragments, etc).
  • Example 1 Construction of a Multi-Emission Fluorescent-Labelled DNA ladder (MEFL DNA standard)
  • the MEFL DNA ladder reagent according to the present invention was prepared as follows:
  • OLIGO 1 ⁇ 5'-(ROX)(C6-NH)CTCCTTCTCTCCTT(dT- FAM)CTTTCTCCTCTTTC(dT-C6-NH-TAMRA)TCCTC-3' ⁇ (SEQ ID No. 1) where ROX is the single isomesr of carboxy-X-rhodamine linked to a 5 prime C6 amine group, dT-FAM is fluorescein linked to the C5 position of thymidine, and dT-TAMRA is 6- Tetrame ylrhodarn e-5(6)-carboxamide attached to the C6 of thymidine.
  • the multi-emission fluorescent-labelled (MEFL) polynucleotide, OLIGO 1 was synthesized by automated DNA synthesis protocols using standard phosphoramidite chemistry by TriLink BioTechnologies, Inc. (San Diego, CA, USA), OLIGO 2 (5'-GATCGAGGAAGA GAGGAGAAA ' 3') (SEQ ID No, 2), which is partially complementary to OLIGO 1, was synthesized by automated DNA synthesis protocols using standard phosphoramidite chemistry by Sigma-Aldrich (St Louis, MO, USA).
  • a double-stranded DNA linker that could be ligated to DNA fragments bearing five prime GATC single stranded overhang was created by annealing 30 pmol of OLIGO 1 and 60 pmol of OLIGO 2 in 10 ⁇ l of lx Hybridisation Buffer (100 mM ' sodium chloride; 2 mM EDTA). To aid the annealing process, the ohgonucleotides were heated to 70°C for 5 min in a heated water bath. The water bath was switched off and the hybridisation reaction allowed to cool slowly to room temperature over 30 min.
  • the plasmid pGEM3Zf(+) (SEQ ID No. 3) was digested with the restriction enzyme _5i. «3A.
  • the digestion reaction contained 1 ⁇ g pGEM3Zf(+), 1 ⁇ l lOx Buffer 1 (lOO mM Tris-HCl; 100 mM magnesium chloride; 10 M dithiothreitol, pH 7.0 at 25 "C), 3 units of SatiSh (New England Biolabs, Beverly, MA), and sterile molecular biology grade water to a final volume of 10 ⁇ l.
  • the reaction was incubated at 37°C fo ⁇ 2 h before the Sau A restriction enzyme was heat inactivated at 70"C for 20 min.
  • the digested DNA was precipitated by addition of 10 ⁇ l of 5 M potassium acetate (pH5.2), 1 ⁇ l of 20 mg/ml glycogen (Sigma-Aldrich), 90 ⁇ l of sterile molecular biology grade water, and 100 ⁇ l of 100% isopropanol.
  • the sample was incubated on ice for 10 min before centrifugation at 14,000 g for 10 min. After discardin the supernatant, the resulting DNA pellet was washed three times with 70% ethanol, air dried, and resuspended in 10 ⁇ l of TE buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA, pH 8).
  • the MEFL polynucleotide was ligated to the Sau3A digested pGEM3Zf(+) plasmid.
  • the ligation reaction contained: 200 ng of S ⁇ 3A digested ⁇ GEM3Zf(+), 1 ⁇ l lOx ligation buffer (500 mM Tris-HCl, pH 7.8; 5 mM adenosine triphosphate 100 mM beta-mercaptoethanol; 50 mM MgCl 2 ).3 ⁇ l of 50% PEG 8000 (Sigma-Aldrich), 1.5 Weiss units of T4 DNA ligase (Promega, Madison, WI), and sterile molecular biology grade water to a final volume of 10 ⁇ l. All components except the PEG and ligase were added and mixed before the PEG 8000 was added. The PEG was mixed thoroughly with the other components before the T4 DNA ligase was added.
  • the ligated MEFL DNA was precipitated by addition of 100 ⁇ l of a 50% w/vol PEG 8000 solution and centrif ⁇ ging at room temperature at 14,000 g for 10 min. The supernatant was removed and the pellet resuspended in 10 ⁇ l of TE buffer.
  • the MEFL ligated DNA was digested in separate reactions with the restriction enzymes AM and Mspl to generate two MEFL DNA ladders.
  • the first digestion reaction contained: 40 ng of ligated DNA; 1 ⁇ l lOx Buffer 2 (100 mM Tris-HCl, pH7.9 at 25 * C; 100 mM magnesium chloride; 500 mM sodium chloride; lO M dithiothxeitol); 5 units of AM (New England Biolabs, Beverly, MA), and sterile molecular biology grade water to final volume of 10 ⁇ l.
  • the reaction was incubated at 37'C for 2 h before the M ⁇ estrietion enzyme was heat inactivated at 70°C for 20 min.
  • This MEFL DNA ladder is hereby referred to as the "Sau3A/AM MEFL DNA ladder" or "Sau3A/Alul MEFL DNA standard”.
  • a second digestion reaction was performed that contained: 40 ng of ligated DNA; 1 ⁇ l 10x Buffer 2 (100 mM Tris-HCl, pH7.9 at 25'C; 100 mM magnesium chloride; 500 mM sodium chloride; lOmM dithiothreitol); 10 units of Mspl (New England Biolabs, Beverly, MA), and sterile molecular biology grade water to a final volume of 10 ⁇ l.
  • the MEFL DNA ladders were precipitated by addition of 10 ⁇ l of 5 M potassium acetate (pH5.2), 1 ⁇ l of 20 mg ml glycogen (Sigma-Aldrich), 90 ⁇ l of sterile molecular biology grade water, and 100 ⁇ l of 100% isopropanol to each reaction.
  • the samples were incubated on ice for 10 min before cenlrifugation at 14,000 g for 10 min. After discarding the supernatant, the resulting DNA pellets were washed 3 times with 70% ethanol, air dried, and resuspended in 10 ⁇ l of TE buffer.
  • the resulting chromatographic data were processed using Algorithm 1 to determine the relative signal contribution of the MEFL DNA standards.
  • the MEFL DNA standard described in Example 1 generated a relative signal contribution of 1.00, 2.58, 1.45, and 4.53 in the A, C, G, T channels, respectively.
  • a representative chromatographic trace, generated from 2 ng of the Sat ⁇ AJMspl MEFL DNA standard, is shown in Figure 1.
  • This example demonstrates the identification and removal of MEFL DNA standards from four channel, single lane automated DNA sequencing chromatogcaph data.
  • a series of DNA sequencing reactions of the ⁇ GEM3Zf(+) (SEQ ID 3) plasmid were performed using: 1 ⁇ l (200 ng) of pGEM3Zf(+); 5 pmol of the (-21) M13 Forward primer (5'- GTAAAACGACGGCCAG-3'; SEQ ID No.4) (Applied Biosystems); 2 ⁇ l of 4M trimethylamide N-oxide (Sigma-Aldrich); 1 ⁇ l 2.5x dilution buffer (200 mM Tris-HCl, pH9.0; 5 mM magnesium chloride), 1 ⁇ l of BigDyeTM vers.1 (Applied Biosystems), and sterile molecular biology grade water to a final volume of 8 ⁇ l.
  • the sequencing reactions were performed using a PE9700 thermal cycling machine using the following cycle conditions: one cycle of 96"C for 30 s, followed by 40 cycles of 96'C for 10 s, 50°C for 5 $ and 60'C for 4 rnin.
  • the sequencing reactions were transferred to 0.5 ml tubes and excess labelled dideoxy ⁇ ucleotides removed by phenol extraction and butanol precipitation (Tillett et 5 al.,1999s).
  • 10 ⁇ l of sterile molecular biology grade water and 20 ⁇ l 50:49:1 ⁇ henol:chlorofo ⁇ n:isoamyi alcohol (Sigma-Aldrich) was added before vortexing for 15s.
  • the samples were centrifuged at 14000 g for 3 min.
  • the aqueous layer was transferred to a new 0,5 ml tube containing 180 ⁇ l 100% n-butanol (Lab- Scan, Bangkok, Thailand).
  • the samples were vortexed for 15s before centrifugation at 10. 14 OOOg for 10 min. Without disturbing the pellet, the supematants were discarded and the samples centrifuged for 1 min to remove the remaining supernatant. The DNA pellets were then dried at 80"C for 20 min.
  • the remaining pGEM3Zf(+) sequencing reaction pellets were resuspended in 1 ⁇ l of a serial dilutions (2 ng, 1 ng, 0.5 ng, 0.25 ng or 0.125 ng in lx Loading Buffer) of 20 either the SauiAJAlul or the $au3A/MspI MEFL DNA ladders.
  • the resuspended samples were heat denatured at 90°C for 2 min, placed on ice and loaded onto the ABI377 sequencing gel.
  • the chromatographic data was collected using filter set E and the supplied BigDyeTM vers.l crosstalk matrix using the Sequence Analysis 3.4.1 software (Applied Biosystems) package supplied with the DNA sequencer.
  • FIG. 1 shows a region of the chromatographic trace data resulting from the addition of 2 ng of the SauiAfMspl MEFL DNA standard to the pGEM3Z(f+) DNA sequencing reaction.
  • the trace data has been baselined and crosstalk corrected using the supplied BigDye vers.1 matrix.
  • a single peak derived from the MEFL DNA standard (with signal in all four channels) can be seen at approximately scan number 3500. This peak has been indicated with an arrow.
  • Figure 2B shows the same region after processing the trace data using the steps described hx Algorithm 2.
  • a single pealc can be observed at approximatel scan number 3500 indicating the presence of a MEFL standard peak at this location. This demonstrates that a MEFL DNA standard can be identified and the peak modelled even in the presence of signal in all four emission channels.
  • the emission signal derived from the MEFL DNA standards must be removed.
  • the MEFL DNA standard cause a "corruption" of the sequence data by contributing signal across all four channels, preventing the bases sequence from being accurately determined.
  • the sequence of the region between scan numbers 3450 and 3560 was determined to be CTGCNCTCGG, where N was indeterminate due to signal derived from the MEFL DNA standard.
  • the location of the deter inate position is indicated by an arrow in Figure 2A.
  • Removal of the MEFL DNA standard signal can be performed by following the steps described in Algorithm 3, in combination with the data derived from Algorithms 1 and 2.
  • Figure 2C show the resulting chromatographic data after application of Algorithms 1, , and 3, followed by mobility shift correction and inter-channel normalisation.
  • Mobility shift correction and inter-channel normalisation are techniques well known to the art and are described in Giddings et al (Giddings et al. 1993; Giddings et al. 1998).
  • Giddings et al. 1993; Giddings et al. 1998 At the prior location of the MEFL DNA standard peak, only one major peak is retained (in the G channel). This is indicated by an arrow.
  • This example describes the calibration of the MEFL DNA standards by use of the ROX 500 internal DNA size marker.
  • a calibration process may be used to ensure that any linear or non-linear mobility effects arising from multiple fluorescent labelling can be identified. Any such mobility effects may be incorporated into fragment sizing models to ensure accurate absolute fragment sizes are obtained.
  • the SauiA/Alul and SauSAlMspl MEFL DNA ladders were separately rnixed with the intemal-lane size marker GENESCAN-ROX 500 (Apphed Biosystems) that has fragment molecular lengths of 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500 nucleotides.
  • Each sample contained 0,5 ⁇ l of GENESCAN-ROX 500 and 2 ng of either the Saul Al AM or SauS lMspl MEFL DNA ladders in 1 ⁇ l of lx Loading Buffer. Samples were loaded onto a sequencing gel and run on an ABI PRISM 377 DNA sequencer (Applied Biosystems). Chromatograph data was collected using filter set E and processed using the Sequence Analysis 3.4.1 software package (Applied Biosystems).
  • Figure 3A shows the region of the trace from scan number 0 to 910. Two peak types were observed: 1. Peaks that fluoresced equally in the C and T channels (ROX), 2, Peaks that fluoresced in all four channels (MEFL DNA standard). The ROX derived peaks are marked with a * (star) and the MEFL standard derived peaks with arrows.
  • the chromatograph trace data was processed using Algorithm 2 and the MEFL standard relative emission contributions calculated in Example 1.
  • the section of the resulting trace corresponding to scan numbers 0 to 910 is shown in Figure 3B.
  • MEFL DNA standard peaks were observed at scan numbers 62, 71, 166, 508, and 763.
  • the chromatograph trace data was further processed using Algorithm 3 and the MEFL DNA standard peak location calculated previously.
  • ROX derived peaks were observed at scan numbers 10, 191, 383, 90, 795, and 867 in this section of the trace file, corresponding to the 50, 75, 100, 139, 150 and 160 nucleotide ROX size markers ( Figure 3C). These peaks were used to generate a linear best-fit model y- 0 273 ⁇ + SO.042, where y is size in nucleotides and x is scan number) that could be used to provide a sizing estimate of the MEFL DNA standard peaks (Table 1). This model predicted a size of 58, 59, 71, 115, and 147 nucleotides fbr the five measured MEFL DNA standard derived peaks shown in Figures 3A and 3B.
  • This example describes the use of the MEFL DNA standard to size PCR amplified microsatellite fragments.
  • the SaitBAJMspl MEFL DNA ladders were separately mixed with the PCR amplified microsatellite containing the internal-lane size marker GENESCAN- TAMRA 500. Each mix contained 2 ⁇ l of the FCR/GENESCAN-TAMRA 500 solution and 2 ng of S u3A/Ms ⁇ MEFL DNA ladders in 1 ⁇ l of lx Loading Buffer. One icrolitre of these samples was loaded onto a sequencing gel and run on an ABI PRISM 377 DNA sequencer (Applied Biosystems). Chromatograph data was collected using filter set E and processed using the Sequence Analysis 3.4.1 software package (Applied Biosystems).
  • Figure 4A shows the region of the trace from scan number 1480 to 3440.
  • Three peak types were observed: 1. Peaks that fluoresced equally in the C and A channels (TAMRA). 2, Peaks that fluoresced in all four channels (MEFL DNA standard). 3. Peaks that fluoresced in the G channel (microsatellite).
  • TAMRA derived peaks are marked with a * (star), the MEFL standard derived peaks with arrows, and the two microsatellite peaks with PI and P2 ( Figure 4A).
  • the cltfomalograph trace data was processed using Algorithm 2 and the MEFL standard relative emission contributions calculated as in Example 1.
  • MEFL DNA standard peaks were observed at scan numbers 1054, 1307, 1328, 1342, 1541 and 3347 corresponding to the 182, 205, 207, 209, 227, 394 nucleotide sized fragments.
  • the section of the resulting trace corresponding to scan numbers 1480 to 3440 is shown in Figure 4B, In this section of the trace, MELF derived peaks were observed at scan numbers 1541 and 3347 (indicated with arrows), corresponding to the 227 and 394 nucleotide MELF size standard markers ( Figure 4B).
  • This model predicted a size of 253.74 and 282.11 nucleotides for PI and P2 respectively. These values match within experimental error the peaks size previously calculated for these fragments of 253.30 and 281.88 nucleotides using the commercially available GENESCAN software.
  • the ideal multi-channel emission spectra for use with Algorithm 1 has an equal signal intensity across all four data channels.
  • the relative channel signal of a given MEFL polynucleotide will vary depending on the fluorescent dye combination and DNA sequencer used.
  • the MEFL polynucleotide described in Example 1 (OLIGO 1) does not emit equal signal intensity in all four data channels when used with the ABI377 or ABI3730 DNA sequencers.
  • OLIGO 5 was synthesized by automated DNA synthesis protocols using standard phosphoramidite chemistry by TriLink Bi ⁇ Technoiogies, Inc. (San Diego, CA, USA). Exa ple 7: Comparison of the relative signal contributions of OLIGO 1 and OHGO 5 when analysed on an ABI 377 DNA sequencer.
  • OLIGO 5 SEQ JD No. 5 described in Example 6
  • ABI PRISM 377 automated DNA sequencer Applied Biosystems, Foster City, CA, USA
  • a dilution series of OLIGO 5 were analysed.
  • Each lane contained 100 finol, 51 finol, 25.5 fmol, 12,75 fmol, 6.3 fmol, 3.1 fmol or 1.5 finol of OLIGO 5 in 1 ⁇ l of lx Loading Buffer (95% vol/vol deionized formamide; 5% vol/vol 0.5 M EDTA and 50 mg/ml dextran blue).
  • the chromatographic data was collected using filter set E and the 377XL DNA Sequencer Data Collection version 2.0 software packages (Applied Biosystems).
  • the resulting chromatographic data files were analysed using the Sequence Analysis 3.4.1 software package (Apphed Biosystems).
  • the relative signal contributions of the OLIGO 5 were determined by measuring the peak heights from the observed channel baseline.
  • OLIGO 5 provided a relative signal contribution of 1.1, 2.0, 1.0, and 2.5 in the A, C, G and T channels, respectively.
  • Oligo 1 provided a relative signal contribution of 1 ,5, 2.8, 1,0, abd 3.5 in the A, C, G, and T channela, respectively.
  • a representative peak from a chromatographic trace, generated from 51 fmol of the MEFL polynucleotide, OLIGO 1, and 100 finol of the MEFL polynucleotide, OLIGO 5, is shown in Figure 6. It was found that, when analysed on an ABI377 DNA sequencer, the relative signal contributions from OLIGO 5 (B in Figure 6) are in a narrower range than those from OLIGO 1(A in Figure 6). In particular, it can be seen from Figure 6 that the signal for the G channel increases relative to the other channels when OLIGO 5 is used.
  • Example 8 Relative signal contributions of the OLIGO 1 and OLIGO 5 when analysed on an ABI3730 automated DNA sequencer.
  • the relative signal contribution of the MEFL polynucleotide, OLIGO 1 (Example 1), and the MEFL polynucleotide OLIGO 5 (Example 6) was investigated on an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Samples comprising 127 finol, 63 finol, 31 fmol, 15 frnol, 7.5 finol or 3.7 fmol of OLIGO 1 or 63 fmol, 31 fmol, 15 f ol, 7.5 fmol, 3.7 finol or 1.8 finol of OLIGO 5 in 10 ⁇ l of deionized water were prepared and injected onto the ABI3730 DNA sequencer. The chromatographic data was collected using the DT3730POP7(BDv3).mob crosstalk matrix (Applied Biosystems) supplied with the automated DNA sequencer.
  • the resulting chromatographic data files were analysed using the Sequence Analysis 3.4, 1 software package (Applied Biosystems).
  • the relative signal contributions, of OLIGO 1 and OLIGO 5 were determined by measuring the peak heights from the observed channel baseline.
  • the MEFL OLIGO 5 generated a relative signal contribution of 1.0, 1.3, 1.0, and 3.0 in the A, C, G, T channels, respectively.
  • the MEFL polynucleotide OLIGO I generated a relative signal contribution of 1.3, 1.6, 1.0, and 4,0 in the A, C, G, T channels, respectively, (It should be noted that the relative channel signal of a given MEFL polynucleotide is machine specific due to the spectral calibration.)
  • This example demonstrates that the signal intensity of the MEFL polynucleotide can be controlled by variation of the spacing between the fiorescent moieties. This allows the MEFL polynucleotide to be adapted to specific instruments and applications.
  • Mitchelson K.R.2001. The application of capillary electrophoresis for DNA polymorphism analysis. Methods Mol Biol 162: 3-26. Mitnikj L., M, Novotny, C. Felten, S, Buonocore, L. Koutny, and D. Schmalzing.2001.

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Abstract

L'invention concerne des procédés de détermination de la taille de fragments de polynucléotides au moyen d'une détection d'émission au scanner fluorescent de jeux d'échantillons multiples, étiquetés par fluorescence par électrophorèse, et de marqueurs de dimensions étiquetés par fluorescence, qui sont amenés dans les mêmes voies que les jeux d'échantillons. Les étalons de masse moléculaire possèdent des capacités d'émission fluorescente de longueurs d'onde multiples qui peuvent être détectées, à plus d'une longueur d'onde d'émission, par le scanner fluorescent. Au moins une longueur d'onde d'émission fluorescente desdits étalons est la même que la longueur d'onde d'émission fluorescente d'au moins l'un des jeux d'échantillons. Ceci permet de traiter davantage d'échantillons en parallèle, du fait qu'on élimine l'exigence pour un mode d'exploration/mode de détection de longueur d'onde du détecteur d'émission/scanner fluorescent uniquement destiné à l'étalon.
PCT/AU2003/001640 2002-12-09 2003-12-09 Reactif et procede de determination de la taille de polynucleotides WO2004053153A1 (fr)

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Publication number Priority date Publication date Assignee Title
JP2018523115A (ja) * 2015-07-01 2018-08-16 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ 生体分子のサイズを決定するための方法
US11237130B2 (en) 2015-07-01 2022-02-01 Cytiva Sweden Ab Method for determining a size of biomolecules

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