WO2000036414A1 - Methods and products for peptide-based dna sequence characterization and analysis - Google Patents
Methods and products for peptide-based dna sequence characterization and analysis Download PDFInfo
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- WO2000036414A1 WO2000036414A1 PCT/US1999/030104 US9930104W WO0036414A1 WO 2000036414 A1 WO2000036414 A1 WO 2000036414A1 US 9930104 W US9930104 W US 9930104W WO 0036414 A1 WO0036414 A1 WO 0036414A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions
- This invention relates to the fields of Molecular Biology and Genetics, with particular reference to the identification and analysis of DNA molecules. BACKGROUND
- the fragment may be derived from genomic DNA of viral, procaryotic or eucaryotic origin, or it may be a derived from cDNA. In many cases, the fragment derives from a larger DNA molecule, or set of molecules, whose sequence
- a number of methods presently exist for determining the nucleotide sequence of a DNA fragment involves cloning the fragment in a plasmid vector of known sequence, purifying the plasmid DNA, annealing a primer complimentary to a portion of the known sequence to one strand of the molecule, extending the primer with DNA polymerase, terminating the polymerization with dideoxy nucleotides, and comparing the lengths of the various terminated molecules to reveal the nucleotide sequence 3' to the primer.
- SSCP single strand conformational polymorphism analysis
- EMD nuclease analysis
- ASO allele-specific oligonucleotide hybridization
- an exon is assayed for chain termination mutations by PCR-amplifying the exon, expressing it in a cell free transcription/translation system, and examining the expressed polypeptide by SDS polyacrylamide gel electrophoresis to determine if it is smaller than a non-mutant control polypeptide. While the protein truncation assay can reveal the presence of a nonsense or frameshift mutation, it is important to note that the assay does not reveal the molecular nature or exact location of the mutation - one does not know if it is a TAG, TGA, TAA or frameshift mutation, and one only knows the approximate location of the mutation within the exon.
- the database search activities in the prior art are aimed at protein identification and/or analysis
- the search activity is aimed at DNA identification or analysis.
- the artificial hybrid peptides that are analyzed in the instant invention are not naturally occurring, nor are they necessarily biologically active. And yet they have distinct utility as reporters that carry information about the nucleic acids that encode them.
- peptide reporters provides a number of clear advantages over analysis of the DNA sequences that encode them.
- One advantage derives from the fact that a peptide is considerably smaller than the DNA that encodes it (individual amino acids averages about 110 Da each whereas the trinucleotides (triplets) that encode them average over N Daltons each) .
- Another advantage derives from the fact that peptides are much more diverse in composition than nucleic acids, as they are composed of combinations of 20 different amino acids instead of combinations of 4 different nucleotides.
- the DNA to be analyzed is incorporated into a hybrid artificial gene that is then transcribed and translated to produce a hybrid peptide. Analysis of the peptide, rather than analysis of the DNA, is used to gain sequence data about the DNA.
- the mass and/or composition and/or partial or complete amino acid sequence of the hybrid peptide is determined, and the data are used to search for matches in data sets produced by in silico transcription and translation of hybrid artificial genes created in silico using the reference sequence, or using transformations of the reference sequence such as single nucleotide deletions or substitutions thereof.
- This peptide-based approach to DNA sequence-determination is fundamentally different from all other methods in the art, none of which employs transcription, translation and peptide analysis, as does the instant invention.
- the invention depends on means to translate a portion of the unknown sequence as part of a fusion peptide whose synthesis originates in the known sequence and extends into the unknown sequence that is being characterized.
- the unknown sequence need not comprise actual protein-coding sequence in the cell from which it originates, although it may in some cases, and so the invention is of general applicability and not confined to coding sequences.
- the invention also depends on means to accurately measure the mass and/or composition and/or partial or complete amino acid sequence of the fusion peptide. Many methods for making such measurements are known in the art, and a number of them will be discussed later in this specification. But first, let us consider the issue of the expected sizes, masses, and amino acid sequences of the peptides that can be translated from an unknown sequence.
- the likelihood that the first codon in the sequence is a nonsense codon (and that the peptide will thus be zero amino acids in length) is 1/21, or -4.7%.
- the likelihood that the first codon is not a nonsense codon and the second codon is a nonsense codon (and that the peptide will thus be one amino acid in length) is 20/21 x 1/21, or -4.5%.
- the likelihood that the first and second codons are not nonsense codons and the third codon is a nonsense codon
- the table below shows the calculated probabilities, for the first 24 codons of a random DNA sequence, that a given peptide will be of a given length or less.
- the table indicates that, for example, 0.705
- the probability that a sequence of a given length translated from it will have a particular amino acid sequence can be calculated simply by multiplying together the frequencies in the genetic code of the codons encoding each amino acid amino acid in the sequence. Since some amino acids have as many as six codons and others as few as one, the predicted frequency will vary depending on the amino acid sequence itself. Thus the sequence LRRLLR, made up entirely of six-codon amino acids, will appear at a frequency of 1 in (6/61)6, or approximately once in one million codons, and the sequence MWWMMW, made up entirely of one-codon amino acids, will appear at a frequency of 1 in (1/61)6, or approximately once in fifty billion codons.
- the number of distinct amino acid combinations and their frequencies is represented by the polynomial expansion (a+b+c+d+ +q+r+s)N, where the letters "a" through “s” (19 letters) represent the frequencies in the genetic code of each amino acid (there are 19 instead of 20 letters because two amino acids, leucine and isoleucine, have the same mass and must be treated as a group) and N represents the length of the peptide.
- the number of terms in the expansion represents the number of composition classes, and the value of each term divided by the sum of the values of all of the terms gives the frequency of any given class. It should be clear to the reader that for all but very small values of N, the frequency of any given class will be very low.
- the operation of the invention depends upon the presence of a specially engineered DNA sequence adjacent to the unknown DNA.
- the engineered sequence contains at minimum the following elements: (1) a promoter sequence oriented to promote transcription into the unknown sequence, and (2) a translation initiation sequence, and
- a coding sequence comprises at minimum a start codon. Transcription from the promoter, followed by translation of the transcript beginning at the start codon, yields a fusion peptide with an N-terminal portion of known amino acid composition followed by a portion of unknown sequence encoded by the unknown DNA. A second known sequence may, in some embodiments, be incorporated into the C-terminal portion of the fusion peptide. Analysis of fusion peptides
- a fusion peptide Once a fusion peptide has been produced as described above, it must be analyzed to determine its mass and/or its composition and/or its amino acid sequence.
- Mass Spectrometry is one preferred analytical method because it is fast and highly accurate. A number of specific examples of the application of mass spectrometric analysis to fusion peptides are given later in this specification.
- the data are compared with the data set generated in silico that contains all possible fusion peptides generated by fusing the known sequence to the reference sequence at all possible positions in the reference sequence and calculating the masses and/or compositions and/or amino acid sequences of the resulting peptides.
- IMAC insulin-binding protein
- GST glutathione-S-transferase
- MBP maltose binding protein
- GST glutathione-S-transferase
- MBP maltose binding protein
- Many means for separating and/or purifying peptides or proteins are also well known and may be applied in certain embodiments of the invention.
- Certain embodiments of the invention can be used to detect and characterize naturally occurring mutations and DNA polymorphisms, including single nucleotide polymorphisms (SNPs) . This is done by comparing the coding capacity of subsets of the reference sequence with the coding capacity of equivalent subsets of the sequence derived from it by specific nucleotide changes, as follows.
- SNPs single nucleotide polymorphisms
- coding capacity is meant the set of the amino acids encoded in at least one reading frame of a sequence; a change in the coding capacity would be due, at minimum, to a change in amino acid composition of at least one encoded peptide.
- an additional related set of peptides is generated by generating, also in silico, a set of transformed DNA sequences derived from the same portion of the reference DNA sequence, each member of the set containing a different sequence alteration. Each member of the transformed set is then translated in silico to give a transformed set of peptide sequences.
- the expanded set of peptides will contain 3N members, where N is the length of the relevant portion of the reference nucleotide sequence. (In most cases, some of the members of the new set will be identical due to the degeneracy of the genetic code.)
- N is the length of the relevant portion of the reference nucleotide sequence.
- mutations or DNA polymorphisms are detected and quantified, by first producing a PCR amplicon representing a distinct portion of the reference sequence, such as a single exon in a gene of interest.
- the amplicon is expressed as part of a fusion peptide as described previously.
- the exon is expressed in frame with respect to the translation initiation codon in the vector, with the result that the peptide comprises the entire amino acid sequence encoded in the exon.
- the PCR template contains a point mutation that alters the amino acid sequence, this will be observed as, for example, a distinct change in the mass of the peptide relative to the mass of the peptide from the non-mutant exon.
- a large number of diseases are known to be caused by mutations in known genes, and the mutations in these genes that are responsible for dominant or recessive genetic disease may be examined using the instant invention. These include: Ataxia talangietasia (ATM),
- Familian adematous polypsosis APC
- Hereditary breast/ovarian cancer BRCA1, BRCA2
- Hereditary melanoma CDK2, CDKN2
- the EMBL3 clone HG3 contains a 10942 base pair insert containing the human nucleolin gene as well as surrounding intergenic sequences (Srivistava, Genbank accession number gb J05584) .
- Purified HG3 DNA is digested to completion with the restriction endonuclease EcoRI and a plasmid mini-library is constructed by cloning the fragments into the EcoRI site of the vector pUC19 using standard methods.
- the library is transformed into competent E. coli BLR cells. Ampicillin resistant colonies are selected on LB ampicillin plates, and a single colony is picked and used to prepare a plasmid miniprep .
- a 250 ml liquid culture of cells from this colony is grown in LB-ampicillin medium at 25 degrees to a density of 2 x 108 cells per ml, induced with 1 mM IPTG for 2 hours, concentrated to a volume of 10 ml by centrifugation, and lysed by sonication in the presence of the protease inhibitors AEBSF, bestatin, E-64 and pepstatin A.
- a second 250 ml control culture with nonrecombinant pUC19 vector is prepared in parallel. All of the above steps follow standard methods well known in the art.
- a 10 ul aliquot of each cell lysate is subjected to capillary liquid chromatography (LC) followed by electrospray ionization mass spectrometry (ESI/MS) using methods and procedures well known in the art .
- LC capillary liquid chromatography
- ESI/MS electrospray ionization mass spectrometry
- the spectrum of the lysate from the induced cells is observed to contain a distinct peak, at a position corresponding to a mass of 5253_ 2_Daltons that is not observed in the control cell lysate.
- the J05584 sequence is scanned to identify each EcoRI site. Five such sites are identified. Each EcoRI fragment is ligated, in silico, to the EcoRI site in the pUC19 vector, producing 10 possible recombinant plasmids, one for each of the two possible orientations of each insert in the vector.
- the predicted amino acid sequence and molecular mass of each IPTG- inducible hybrid translat ion product ( translated from the mRNA transcribed from the lac promoter in the vector) is calculated , and the masses of the ten possible polypept ides are tabulated , as shown in the table below . Position of EcoRI site Orientation in pUC19 Predicted Peptide Mass
- the starting material was a cloned gene. If one begins instead with a cloned a cDNA library and uses identical procedures in an iterative manner, the identity of multiple members of the library are ascertained.
- Example 2 Identification of a subcloned EcoRI fragment of a cloned human gene using peptide affinity capture.
- the peptide TMITPSLHACRSTLED representing the N-terminal 16 amino acids of the alpha-complementing factor of beta-galactosidase encoded in pUC19 (and also representing the 16 constant N-terminal amino acids in all of the peptides described in Example 1 above) is used to raise a polyclonal rabbit antibody using standard procedures .
- Example 2 A single ampicillin resistant E. coli colony derived from the mini -library transformation described in Example 1 is picked and induced lysates are prepared as described in Example 1.
- a control lysate from cells with nonrecombinant vector is prepared in parallel .
- Immunoreactive proteins are precipitated from the lysates by incubation of 1 ml aliquots with a 1:100 dilution of antiserum followed by precipitation with Protein-A using standard methods.
- the immunoprecipitate is suspended in 50 ul H20, and a 10 ul aliquot is suspended in 40 ul of MALDI-matrix (_-cyano-4-hydroxycinnamic acid dissolved in 1:2 acetonitrile : 1.5% trifluoroacetic acid (ACCA) , and 100 nL applied to the MS probe, air dried, and subjected to matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry using methods and procedures well known in the art.
- MALDI-matrix _-cyano-4-hydroxycinnamic acid dissolved in 1:2 acetonitrile : 1.5% trifluoroacetic acid (ACCA)
- ACCA trifluoroacetic acid
- the mass spectrum of the immunoprecipitate from the induced cell lysate of the clone under examination is observed to contain a distinct peak, at a position corresponding to a mass of 8485_3 Daltons, that is not observed in the control .
- Comparison of the experimental results with the values in the table in example 1 above indicates that the insert begins at position 9241 of the reference sequence and proceeds from left to right in the Genbank sequence .
- Retrieval of the reference sequence beginning at position 9241 indicates that the cloned sequence begins with "GAATTCACATAAATCGCAAATTTTTTTTTCCTTCCC AGAGCCATCCAAAACTCTGTTTGTCAAAGGCCTGTCTGAGGATACCACTGAAGAGAC ATTAAAG" (first 100 nucleotides shown). The identification is confirmed by dideoxy sequencing as described in Example 1.
- Example 3 Identification of a subcloned EcoRI fragment of a cloned human gene: analysis of peptides from multiple reading frames .
- the vector pTriplEx is digested with the restriction endonuclease Bglll and the ends of the linearized plasmid are backfilled using Klenow fragment of E. coli DNA polymerase I.
- the plasmid is treated with the restriction endonuclease Smal , blunt end ligated with DNA ligase and transformed into competent E. coli BLR cells. Ampicillin resistant colonies are selected on LB ampicillin plates, and a single colony is picked and used to prepare a plasmid miniprep.
- the plasmid here named pTriplEx'
- pTriplEx' is linearized with EcoRI and a mini library is prepared using as inserts the set of fragments produced by complete digestion of the insert in EMBL3 human nucleolin clone described in example 1.
- Competent E coli TOPP-1 cells are transformed with the mini library and a single ampicillin resistant colony is isolated.
- a 250 ml liquid culture of cells from this colony is grown in LB-ampicillin medium at 25 degrees to a density of 2 x 108 cells per ml, induced with 1 mM IPTG for 2 hours, concentrated to a volume of 10 ml by centrifugation, and lysed by sonication on ice with six intermittent 30 second sonication pulses.
- Control_cells with nonrecombinant plasmid are prepared in parallel . Immunoprecipitates of both lysates are prepared as in Example 2.
- each EcoRI site in the J05584 sequence is identified and ligated, in silico, to the EcoRI site in the pTriplEx' vector.
- amino acid sequences of the two expected hybrid translation products are calculated.
- the mass of each peptide is calculated and all 10 peptide pairs are tabulated, as shown in the table below. Comparison of the experimental results (i.e., peptides of 4255 and 2635 Da.) with the values predicted in the table indicates that the insert begins at position 4028 of the reference sequence and proceeds in the forward direction.
- Example 4 Identification of a specific mutation in a human gene .
- Two 20 -nucleotide PCR primers - one representing nucleotides 3190-3210 of the nucleolin sequence described previously (the forward primer) and the other representing the reverse complement of nucleotides 4008-4028 (the reverse primer) - are used to generate an 838 nucleotide PCR amplicon using high fidelity thermostabile proofreading DNA polymerase.
- the amplicon is cloned into the pTriplEx' vector described previously, and 1000 transformant colonies from each amplification are pooled to create five bacterial cultures, two derived from the parents and three derived from their offspring.
- Each bacterial culture is treated as described in the previous example to produce five lysates and five MALDI-TOF mass spectra.
- the spectrum from the father shows two prominent peaks at positions corresponding to 6137 and 5707 Daltons. The same peaks are observed for the peptides derived from two of the offspring.
- the mother and the third child show not two peaks but three, two at 6137 and 5707 Da and a new one at 6169 Da.
- the new peak is 32 Da bigger than the 6137 peak, consistent with a change from valine to methionine with respect to the reference sequence.
- Example 5 Identification of a specific mutations in a human gene; analysis of pooled samples.
- reference sequence In this example known portions of the reference sequence are used to design PCR primers, which are then used to generate PCR products that are cloned, expressed in fusion peptides, and analyzed in a parallel fashion.
- the reference sequence predicts a peptide of a particular mass and composition; deviations from the prediction indicate differences in sequence from the reference sequence, in this example single nucleotide polymorphisms.
- Two oligonucleotide primers are synthesized using standard methods.
- CCCGAATTCAGCAGGTAAAAATCAAGG the first ten nucleotides contain an EcoRI site (underlined) and last seventeen nucleotides correspond to the first seventeen nucleotides of exon 2 of the human nucleolin gene.
- GGGGAATTCTTACTCTTCTCCACTGCTAT the last seventeen nucleotides correspond to the reverse complement of the last seventeen nucleotides of exon 2, followed immediately (in the sense orientation of the oligonucleotide) by the stop codon TAA and a sequence that includes an EcoRI site (underlined) .
- Blood is drawn from twenty individuals and PCR amplicons are produced as described in the previous example, using the two primers just described.
- the amplicons are pooled and cloned into the EcoRI site of pUC19 as described in Example 2 above, and the bacterial cultures are treated as described in Example 2 above to produce a single MALDI-TOF mass spectrum derived from all twenty pooled samples.
- the spectrum shows a major peak at 6873_3 Da. , corresponding the predicted mass of the fusion peptide encoded by the exon 2 reference sequence fused to the vector peptide sequence, and two smaller peaks at 6862_3 Da. and 6915_3 Da.
- the amplitude of the 6862 peak is approximately 1/20 of the 6872 peak, and the amplitude of the 6916 peak is approximately 1/40 that of the 6872 peak.
- the -10 Da. shift in the 6862 peak relative to the 6872 peak is that predicted for a single nucleotide polymorphism (SNP) that produces a proline to serine substitution in exon 2 that is already known to exist in the human population at a frequency of approximately 5%, and so it is concluded that in the forty haploid genomes present in the twenty individuals, two copies of this polymorphism are very likely present.
- the +44 Da shift in the 6916 peak indicates an alanine to aspartic acid substitution in exon 2 that was not previously known, and that is present in one copy in the sample of forty haploid genomes .
- the sample was heterogeneous because amplicons from a number of individual individuals were pooled prior to analysis. But the heterogeneity could, in other cases, be intrinsic to a single sample.
- the sample could be a tumor biopsy containing, for example, a mixture of cells that are heterogeneous with respect to mutations in oncogenes or tumor suppressor genes, and so PCR amplification of the oncogene or tumor suppressor gene would yield a heterogeneous amplicon.
- Example 7 Application of a computer program to generate a data set of mass shifts for all possible single nucleotide substitutions in a nucleotide sequence.
- a computer program was written to compute the mass shifts for all single nucleotide substitutions in a nucleotide sequence.
- the program uses the amino acid mass values given in the table below.
- the input to the program is (1) a nucleotide sequence, and (2) a choice by the user of which of the six possible reading frames (3 forward and 3 reverse) to be considered.
- the program translates the input sequence and computes the masses of the encoded peptides. It then generates all possible single nucleotide substitutions of the sequence, computes a new set of peptides, compares them to the original peptide(s), and lists all of the mass differences between the mutant and non-mutant peptides.
- the program output is a listing of the peptide mass changes for all possible single nucleotide substitutions in the input sequence.
- the program then accepts input representing the mass-shift threshold for detection, i.e., the mass shift below which the shift is treated as not detectable.
- Output is a listing of all mutations in the sequence that are not detectable at the set threshold.
- the program was run with the 24 nucleotide input sequence CAACTAGAAGAGGTAAGAAACTAT . Two reading frames were selected; the forward reading frame beginning with the first nucleotide (FI) and the reverse (antisense) reading frame beginning with the second antisense nucleotide (R2) .
- the numbers in the first column denote each nucleotide in the sequence. Note that for each nucleotide in the input sequence there are three possible substitutions, so that the number of lines in the output data set is 72 (3 x 24) .
- the amino acids encoded in each FI codon are shown in the second column, followed by all possible single nucleotide substitutions at each position in the fourth column.
- the fifth column shows the amino acids encoded by the new codons
- the sixth column shows the mass change (if any) due to the amino acid substitution (if any) or translation termination (if any) due to the nucleotide substitution.
- the last column shows the mass changes due to the same substitutions when translation is in the R2 reading frame.
- the detection threshold value of 0.8 Daltons was entered; the program output indicated that only one substitution, at position 1 in the encoded peptide, would go undetected at this threshold value.
- polypeptides from two reading frames makes the analysis significantly more robust than if just one reading frame is used. For example, if just reading frame 1 is used, a shift of -14.03 Daltons could be due to an E-to-D substitution at amino acid 3, or to an E-to-D substitution at amino acid 4, or to an L-to-V substitution at amino acid 2.
- the additional reading frame data are considered, however, each of these possibilities is distinguished from the others and the ambiguity is thereby eliminated. Indeed, when up to six reading frames are considered, there is little or no ambiguity for the great majority of substitutions, even for sequences as long as several hundred nucleotides.
- a data set/database such as that generated above can have great utility in the practice of the instant invention when searched by a computer program that searches the database using experimentally determined peptide mass data. Many such programs can be generated.
- One example is given below. Enter reference sequence
- Example 8 Analysis of exon 2 of the human rds/peripherin gene.
- exon 2 of the human rds/peripherin gene (Genbank accession M73531) is shown below. Intron sequence is shown in lower case; exon sequence in upper case .
- Two primers of sequences GGCCCGGAATTCTCCAGCTGTCTGTTTCCCTTTAAG and AATTTACTCGAGCTACCCCCAGCTGCCCAGGGCCTAC were synthesized and used to PCR amplify rds/peripherin exon 2 from an individual known to carry a wild type allele of rds/peripherin.
- the amplicon was cut with EcoRI and Xhol and cloned into the EcoRI/XhoI sites of the pGEX derivative described in Nelson et al .
- the resulting plasmid was cut with Xho 1, treated with Klenow fragment of DNA polymerase, and self-ligated to produce a construct expected to produce a fusion protein with the sequence shown below.
- the measured masses of the two fusion proteins are 35571 Da and 35630. Da. The difference between the two is 59 Da, indicative of a substitution of arginine for proline in the peptide.
- Examination of the exon 2 sequence reveals a Fin I site (GTCCC) whose last two nucleotides are part of the first proline codon (CCT) in the sequence. It is concluded that a proline-to-arginine substitution is present at this proline. It is further concluded that the codon very likely suffered a transversion at the second position to create the arginine codon CGG. Dideoxy sequencing across the exon 2 sequence in both constructs confirms these conclusions.
- Example 9 In vitro analysis of exon 2 of human rds/peripherin .
- the amplicons described in the previous example are reamplified using the upstream primer 5 ' GGATCCTAATACGACTCACTATAGGGAGACCACCATGCATCACCATCATCACCAT CACCACTCTCCAGCTGTCTGTTTCCCTTTAAG and the downstream primer 5' CTTAGTCATTATACCCCCAGCTGCCCAGGGCCTAC.
- the upstream primer contains a T7 promoter followed by a translation initiation sequence (start codon underlined) followed by a sequence encoding eight histidines followed by sequence identical to the red/peripherin sequence immediately 5' to rds/peripherin exon 2.
- the downstream primer contains two stop codons (in antisense orientation) preceding the sequence complimentary to the sequence just 3' to red/peripherin exon 2.
- the reamplification products are transcribed and translated in a coupled cell free system (transcription by T7 polymerase; translation by rabbit reticulocyte lysate) using established methods and procedures.
- Immobilized metal affinity chromatography is used to purify the translation products, and the translation products are analyzed by MALDI-TOF mass spectroscopy as in the previous example.
- the two major translation products are observed to differ by 59.1 Da, indicative of a substitution of arginine for proline in the polypeptide.
- polypeptide mass the only physical parameter whose value was measured was polypeptide mass. It should be clear to the reader, however, that assessing certain other polypeptide properties, such as amino acid composition or amino acid sequence, may also serve to locate an unknown sequence with respect to the reference sequence. Such data might be obtained, for example, by partial or complete digestion of the peptide, prior to spectrometry, with endopeptidases such as trypsin, chymotrypsin, or pepsin, or with aminopeptidases or carboxypeptidases .
- Analysis can be performed with a variety of spectrometric methods besides MALDI-TOF and ESI, such as tandem mass spectrometry (MS/MS) , quadripole time of flight spectrometry (Q-TOF) , or Fourier transform ion cyclotron resonance (FTICR) mass spectrometry.
- MS/MS tandem mass spectrometry
- Q-TOF quadripole time of flight spectrometry
- FTICR Fourier transform ion cyclotron resonance
- Other analytical methods well known in the art can also be used to analyze the fusion peptides, such as gel or capillary electrophoresis or high performance liquid chromatography (HPLC) . It should also be clear that the instant invention has utility even if it does not unambiguously assign an unknown sequence to just one place in the reference sequence.
- a search might eliminate all but four positions in the reference sequence, each on a different chromosome; if the chromosomal location of the unknown sequence were known from some independent determination, such as fluorescence in situ hybridization (FISH), then the assignment could be made unambiguous.
- FISH fluorescence in situ hybridization
- the reference sequence is complex, representing, for example, an annotated combination of sequences derived from more than one individual, strain or species, which could be viral, procaryotic or eucaryotic.
- the instant invention could be used, in medical, forensic or population biology contexts for example, to determine the individual, strain, or species from which the unknown DNA originated, or, conversely, it could be used to rule out an individual, strain or species as the source of origin of the unknown DNA.
- Some embodiments of the invention include multiplex or pooled-sample analysis wherein peptides encoded in more than one DNA fragment are co-analyzed. For example, peptides encoded in more than one exon of a gene may be combined and analyzed in concert, or samples from multiple individuals may be pooled and analyzed together. Some embodiments of the invention include methods for determining the sequence of a polynucleotide, comprising providing a nucleic acid fragment having homology to a known reference sequence; expressing at least one polypeptide from said fragment; and assessing at least one physical property of said at least one polypeptide to determine the sequence of said fragment by comparing said at least one property to the predicted properties of polypeptides encoded in said known reference sequence.
- the method also includes wherein said nucleic acid fragment contains a difference with respect to the reference sequence wherein said difference is selected from the group consisting of single nucleotide polymorphism, single nucleotide substitution, single nucleotide deletion, single nucleotide insertion, multiple nucleotide substitution, multiple nucleotide deletion, multiple nucleotide insertion, DNA duplication, DNA inversion, DNA translocation, and DNA deletion/substitution.
- said nucleic acid fragment comprises an exon or a cDNA.
- the polypeptide (s) contain heterologous epitope tags and expressed in living cells or expressed in a cell free systems such as an E.
- the invention further includes embodiments wherein the peptides are purified by a variety of methods including gel electrophoresis, capillary electrophoresis, liquid chromatography (LC) , capillary liquid chromatography, high performance liquid chromatography
- LC liquid chromatography
- HPLC differential centrifugation, filtration, gel filtration, membrane chromatography, affinity purification, biomolecular interaction analysis (BIA) , ligand affinity purification, glutathione-S-transferase affinity chromatography, cellulose binding protein affinity chromatography, maltose binding protein affinity chromatography, avidin/streptavidin affinity chromatography, S-tag affinity chromatography, thioredoxin affinity chromatography, metal-chelate affinity chromatography, immobilized metal affinity chromatography, epitope-tag affinity chromatography, immunoaffinity chromatography, immunoaffinity capture, capture using bioreactive mass spectrometer probes, mass spectrometric immunoassay, and immunoprecipitation.
- BIOS biomolecular interaction analysis
- the method further includes embodiments wherein the physical property that is determined is mass, and wherein mass is determined by a variety of methods including mass spectrometry, MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI) ) tandem mass spectrometry (MS/MS) , quadripole time of flight spectrometry (Q-TOF) , Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, gel electrophoresis, capillary electrophoresis, and high performance liquid chromatography (HPLC) .
- the method further includes embodiments wherein the physical property that is assessed is partial or complete amino acid composition or sequence.
- the present invention includes a method for genetic analysis comprising providing a nucleic acid fragment, expressing at least one polypeptide from the fragment, and assessing at least one physical property of said at least one polypeptide to determine the coding capacity of said fragment by comparing said at least one property to the predicted properties of polypeptides encoded in a known reference sequence.
- the invention includes method for analyzing fragments that contain a differences with respect to the reference sequence that include of single nucleotide polymorphisms, single nucleotide substitutions, single nucleotide deletions, single nucleotide insertions, multiple nucleotide substitutions, multiple nucleotide deletions, multiple nucleotide insertions, DNA duplications, DNA inversions, DNA translocations, and DNA deletion/substitutions.
- the invention includes methods for analyzing nucleic acid fragment representing exons or cDNAs , for examining polypeptides that carry epitope tags, for examining polypeptides expressed in living cells or in cell free systems such E.
- the invention further includes embodiments wherein the peptides are purified by a variety of methods including gel electrophoresis, capillary electrophoresis, liquid chromatography (LC) , capillary liquid chromatography, high performance liquid chromatography (HPLC) , differential centrifugation, filtration, gel filtration, membrane chromatography, affinity purification, biomolecular interaction analysis (BIA) , ligand affinity purification, glutathione-S-transferase affinity chromatography, cellulose binding protein affinity chromatography, maltose binding protein affinity chromatography, avidin/streptavidin affinity chromatography, S-tag affinity chromatography, thioredoxin affinity chromatography, metal-chelate affinity chromatography, immobilized metal affinity chromatography, epitope-tag affinity chromatography, immunoaf f inity chromatography, immunoaffinity capture, capture using bioreactive mass spectrometer probes,
- LC liquid chromatography
- HPLC high performance liquid chromatography
- the method further includes embodiments wherein the physical property that is determined is mass, and wherein mass is determined by a variety of methods including mass spectrometry, MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI) ) tandem mass spectrometry (MS/MS) , quadripole time of flight spectrometry (Q-TOF) , Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, gel electrophoresis, capillary electrophoresis, and high performance liquid chromatography
- mass spectrometry MALDI-TOF mass spectrometry
- ESI electrospray ionization mass spectrometry
- MS/MS tandem mass spectrometry
- Q-TOF quadripole time of flight spectrometry
- FTICR Fourier transform ion cyclotron resonance
- the method further includes embodiments wherein the physical property that is assessed is partial or complete amino acid composition or sequence.
- the invention includes methods for assessing a disease, condition, genotype, or phenotype comprising providing a nucleic acid fragment from a biological sample, and expressing at least one polypeptide from said fragment, and assessing at least one physical property of said at least one polypeptide to determine the sequence of said fragment by comparing said at least one property to the predicted properties of polypeptides encoded in a known reference sequence, and correlating said determined sequence with said disease, condition, genotype or phenotype.
- the biological sample may be obtained from a virus, organelle, cell, tissue, body part, exudate, excretion, elimination, or secretion of a healthy, diseased or deceased microorganism, protist, alga, fungus, animal or plant.
- kits for treating diseases, conditions, genotypes, or phenotypes comprising providing a nucleic acid fragment from a biological sample, and expressing at least one polypeptide from the fragment, and assessing at least one physical property of one or more of the polypeptides to determine the sequence of the fragment by comparing the property or properties to the predicted properties of polypeptides encoded in a known reference sequence.
- the sample may be obtained from a virus, organelle, cell, tissue, body part, exudate, excretion, elimination, or secretion of a healthy, diseased or deceased microorganism, protist, alga, fungus, animal or plant.
- the test may detect heterozygote status, and it may indicate responses to drug or therapeutic treatments.
- the test may be for a genetic disease such as
- Alzheimer's disease Ataxia talangietasia (ATM), Familial adematous polyposis (APC) , Hereditary breast/ovarian cancer
- BRCA1, BRCA2 Hereditary melanoma (CDK2, CDKN2), Hereditary non-polypsosis colon cancer (hMSH2 , hMLHl , hPMSl, hPMS2) , Hereditary retinoblastoma (RBI) , Hereditary Wilm' s Tumor (WT1) , Li-Fraumeni syndrome (p53), Multiple endocrine neoplasia (MEN1, MEN2) , Von Hippel-Lindau syndrome (VHL) , Congenital adrenal hyperplasia, Androgen receptor deficiency, Tetrahydrobiopterin deficiency, X-Linked aga maglobulinemia, Cystic Fibrosis (CFTR) , Diabetes, Muscular Dystrophy (DMD, BMD) , Factor X deficiency, Mitochondrial gene deficiency, Factor VII deficiency, Glucose-6-Phosp
- Further embodiments include methods for assessing a disease, condition, genotype, or phenotype providing a nucleic acid fragment from a biological sample, and expressing at least one polypeptide from the fragment, assessing at least one physical property of one or more of the polypeptides to determine the coding capacity of the nucleic acid fragment by comparing said at least one property of the polypeptide (s) to the predicted properties of polypeptides encoded in a known reference sequence, and correlating said determined sequence with said disease, condition, genotype or phenotype.
- the biological sample may obtained from a virus, organelle, cell, tissue, body part, exudate, excretion, elimination, or secretion of a healthy, diseased or deceased microorganism, protist, alga, fungus, animal or plant.
- the particular original source may be blood, sweat, tears, urine, semen, saliva, sweat, feces, skin or hair, or it may come from the environment that the living inhabits or has inhabited, such as air, soil or water.
- Further embodiments include diagnostic or prognostic tests for a disease, condition, genotype, or phenotype selecting a nucleic acid fragment taken from a virus, organelle, cell, tissue, body part, exudate, excretion, elimination, or secretion of a healthy, diseased or deceased microorganism, protist, alga, fungus, animal or plant, expressing at least one polypeptide from the fragment, assessing at least one physical property of the polypeptide (s) to determine the coding capacity of the fragment by comparing the property or properties to the predicted properties of polypeptides encoded in a known reference sequence.
- the particular original source of the nucleic acid may be blood, sweat, tears, urine, semen, saliva, sweat, feces, skin or hair, or it may come from the environment that the living inhabits or has inhabited, such as air, soil or water.
- the test may detect heterozygote status or indicate or response to a therapeutic drug or treatment.
- ATM Ataxia talangietasia
- ATM Familial adematous polyposis
- APC Familial adematous polyposis
- BRCA1, BRCA2 Hereditary breast/ovarian cancer
- BRCA1, BRCA2 Hereditary melanoma
- CDK2, CDKN2 Hereditary non-polypsosis colon cancer
- RKI Hereditary retinoblastoma
- WT1 Hereditary Wilm' s Tumor
- WT1 Li-Fraumeni syndrome
- MEN1, MEN2 Multiple endocrine neoplasia
- VHL Von Hippel-Lindau syndrome
- VHL Congenital adrenal hyperplasia
- Androgen receptor deficiency Tetrahydrobiopterin deficiency
- X-Linked agammaglobulinemia Cystic Fibrosis (CFTR)
- Diabetes
- the invention further includes various polypeptides that are created in the embodiments described above .
- Additional embodiments include computer data structures, comprising: data storage media; and data sets in computer readable form on the data storage media representing a plurality of polypeptide fragments of polypeptides encoded by a reference polynucleotide sequence; and second data sets in computer readable form on the data storage media representing physical properties of each of the polypeptide fragments; and means for correlating empirically derived physical properties of test polypeptides with second data sets to determine the identity of the test polypeptides.
- the data structures may further comprising third data sets in computer readable form on said data storage media representing polynucleotide fragments encoding the polypeptide fragments; and means for correlating the identity of the test polypeptides with polynucleotide fragments represented in the third data sets.
- the physical properties may include mass or partial or complete amino acid composition or sequence.
- the invention includes data structures in which reference polynucleotides have a reading frame, and wherein one data set represents polypeptide fragments encoded in frame and polypeptide fragments encoded out of frame with respect to said reference polynucleotide.
- Further embodiments include computer implemented methods for ascertaining the identity of nucleic acid fragments encoding polypeptides, wherein the nucleic acid fragments are fragments of known reference sequences, comprising the steps of measuring a physical property of a polypeptide comparing, in a computer, the measured physical property with a data set representing the predicted corresponding physical properties of possible polypeptides that are encoded by fragments of the reference sequence within a predetermined size range; and identifying a match between the measured physical property and a predicted physical property in the data set; and displaying or recording the results of the identifying step.
- the data set may includes physical properties of polypeptides encoded by in-frame and any of six out-of-frame fragments of said reference polynucleotide.
- Additional embodiments of the invention include relational data sets useful for detecting and analyzing DNA mutations and polymorphisms comprising a plurality of DNA sequence fragments contained within a reference DNA sequence, the sequences of the polypeptides encoded in said DNA sequence fragments, and the predicted sequences of a plurality of polypeptides encoded in a set of transformed DNA sequence fragments, each member of said set comprised of a DNA sequence related to said DNA sequence fragment by a specific change selected from the group consisting of single nucleotide polymorphism, single nucleotide substitution, single nucleotide deletion, single nucleotide insertion, multiple nucleotide substitution, multiple nucleotide deletion, multiple nucleotide insertion, DNA duplication, DNA inversion, DNA translocation, and DNA deletion/substitution. Further embodiments include computer programs that search of these data sets .
- the computer-implemented methods of the present invention can be carried out on a general purpose computer, such as, for example, a PC running the Windows, NT, Unix, or Linux operating systems, or a Macintosh personal computer.
- a general purpose computer such as, for example, a PC running the Windows, NT, Unix, or Linux operating systems, or a Macintosh personal computer.
- a more powerful computer mainframe would be desirable.
- Suitable computers typically have a central processor, computer memory (such as RAM) , and a storage medium, such as a floppy disk, a fixed disk or hard drive, a tape drive, an optical storage medium such as a CD, DVD, or WORM drive, a removable disk, or the like, which can store data in computer-readable form.
- Such computers typically have a means, such as a monitor, for displaying data or information, and are capable of storing program-generated data in RAM or in the storage medium.
- Such computers can also advantageously be connected to a printer, for providing a fixed record of information generated by the program.
- a general purpose computer utilized in the present invention could be programmed with a specific program of the type described herein.
- this program would generate data sets of all possible nucleotide fragments, in all possible frames and in both orientations. It would predict and store data sets reflecting the translation products of those fragments. It would also store, in a correlatable manner, a data set reflecting a physical property (such as molecular weight) of each of those fragments.
- One program that could be used in the present invention would compare an empirically determined physical property of a polypeptide translated from a polynucleotide fragment from a biological sample with the data set to determine, for example, which possible polypeptide fragment or which possible polynucleotide fragment corresponds to the sample. In this manner, the identity of DNA in the sample can be determined.
- information directly or indirectly related to the identity of the polynucleotide fragment from the sample can be displayed, printed, and/or stored. This can include the exact identity or sequence of the polynucleotide, or a tag, label, or name associated therewith. It could also be a diagnosis of a disease, condition, genotype, or phenotype associated with that particular polynucleotide.
- the invention specified here provides a novel method for analyzing cloned DNA segments and for identifying and/or assaying known or new polymorphisms or mutations in those DNA segments.
- the ' method has unique and highly useful advantages over all other methods the prior art .
- multiple promoters and translation start sites can be placed in the known sequence, on one or both sides thereof, so that the unknown sequence is translated in up to six different reading frames.
- the unknown sequence can be a PCR amplicon that is cloned into a vector in both orientations, thereby yielding a mixture of clones, some translated from one strand and some from the other.
- promoters and translation start signals can be incorporated near one or both ends of a transposable element, such as Tn3 , Tn5 , Tn7 , TnlO, Ty, P-element, and Mariner; of a virus such as herpes virus, adenovirus, adeno-associated virus; or of a retrovirus .
- Fusion protein expression need not take place in bacteria, as in the examples given here, but may take place in eucaryotic cells such as yeast or mammalian cells, and cell free expression need not take place in a rabbit reticulocyte lysate, as in the example, but in other cell free systems.
- peptide capture can be used, such as incorporating biotinylated lysine in the peptides and capturing with avidin or streptavidin.
- protease recognition sites may be incorporated into the known sequence to aid in fragment preparation, such as placing an enterokinase cleavage site and a poly-histidine sequence upstream of the junction to the unknown sequence so that a peptide for analysis can be released by enterokinase treatment of an affinity captured polypeptide.
- DNA polymorphisms that are identified and/or detected need not be limited to single nucleotide polymorphisms, as in the examples, but could be of many other kinds such as microsattelite repeats of different lengths or specific single nucleotide deletions, single nucleotide insertions, multiple nucleotide substitutions, multiple nucleotide deletions, multiple nucleotide insertions, DNA duplications, DNA inversions, DNA translocations, DNA deletion/substitutions or other chromosomal rearrangements .
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Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99968909A EP1151296A4 (en) | 1998-12-16 | 1999-12-16 | Methods and products for peptide-based dna sequence characterization and analysis |
AU27107/00A AU2710700A (en) | 1998-12-16 | 1999-12-16 | Methods and products for peptide-based dna sequence characterization and analysis |
CA002355134A CA2355134A1 (en) | 1998-12-16 | 1999-12-16 | Methods and products for peptide-based dna sequence characterization and analysis |
US09/788,268 US20020155445A1 (en) | 1999-12-16 | 2001-02-16 | Methods and products for peptide based DNA sequence identification and analysis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US11235198P | 1998-12-16 | 1998-12-16 | |
US60/112,351 | 1998-12-16 |
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US09/788,268 Continuation-In-Part US20020155445A1 (en) | 1999-12-16 | 2001-02-16 | Methods and products for peptide based DNA sequence identification and analysis |
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WO2000036414A1 true WO2000036414A1 (en) | 2000-06-22 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US1999/030104 WO2000036414A1 (en) | 1998-12-16 | 1999-12-16 | Methods and products for peptide-based dna sequence characterization and analysis |
Country Status (4)
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EP (1) | EP1151296A4 (en) |
AU (1) | AU2710700A (en) |
CA (1) | CA2355134A1 (en) |
WO (1) | WO2000036414A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105588909A (en) * | 2015-12-15 | 2016-05-18 | 中国肉类食品综合研究中心 | Method for determining multiple kinds of animal origin meat based on liquid chromatographic-tandem mass spectrometric technology |
CN113945649A (en) * | 2021-08-18 | 2022-01-18 | 中国农业科学院农业质量标准与检测技术研究所 | Proteome biomarker of milk with different processing technologies and screening method and application thereof |
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US5620848A (en) * | 1990-06-27 | 1997-04-15 | Trustees Of Princeton University | Methods for detecting mutant p53 |
US5702890A (en) * | 1993-07-26 | 1997-12-30 | K.O. Technology, Inc. | Inhibitors of alternative alleles of genes as a basis for cancer therapeutic agents |
US5876940A (en) * | 1994-11-30 | 1999-03-02 | University Of Utah Research Foundation | Alleles |
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US6017693A (en) * | 1994-03-14 | 2000-01-25 | University Of Washington | Identification of nucleotides, amino acids, or carbohydrates by mass spectrometry |
US6207370B1 (en) * | 1997-09-02 | 2001-03-27 | Sequenom, Inc. | Diagnostics based on mass spectrometric detection of translated target polypeptides |
-
1999
- 1999-12-16 WO PCT/US1999/030104 patent/WO2000036414A1/en not_active Application Discontinuation
- 1999-12-16 EP EP99968909A patent/EP1151296A4/en not_active Withdrawn
- 1999-12-16 CA CA002355134A patent/CA2355134A1/en not_active Abandoned
- 1999-12-16 AU AU27107/00A patent/AU2710700A/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US5620848A (en) * | 1990-06-27 | 1997-04-15 | Trustees Of Princeton University | Methods for detecting mutant p53 |
US5702890A (en) * | 1993-07-26 | 1997-12-30 | K.O. Technology, Inc. | Inhibitors of alternative alleles of genes as a basis for cancer therapeutic agents |
US5876940A (en) * | 1994-11-30 | 1999-03-02 | University Of Utah Research Foundation | Alleles |
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Title |
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LODISH H, DARNELL J, BALTIMORE D: "MOLECULAR CELL BIOLOGY", MOLECULAR CELL BIOLOGY, XX, XX, 1 January 1986 (1986-01-01), XX, pages 107 - 110, XP002926856 * |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105588909A (en) * | 2015-12-15 | 2016-05-18 | 中国肉类食品综合研究中心 | Method for determining multiple kinds of animal origin meat based on liquid chromatographic-tandem mass spectrometric technology |
CN105588909B (en) * | 2015-12-15 | 2020-08-25 | 中国肉类食品综合研究中心 | Method for determining various animal-derived meats based on liquid chromatography tandem mass spectrometry technology |
CN113945649A (en) * | 2021-08-18 | 2022-01-18 | 中国农业科学院农业质量标准与检测技术研究所 | Proteome biomarker of milk with different processing technologies and screening method and application thereof |
CN113945649B (en) * | 2021-08-18 | 2023-08-18 | 中国农业科学院农业质量标准与检测技术研究所 | Proteome biomarker of milk with different processing technologies, screening method and application thereof |
Also Published As
Publication number | Publication date |
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EP1151296A4 (en) | 2005-01-26 |
CA2355134A1 (en) | 2000-06-22 |
EP1151296A1 (en) | 2001-11-07 |
AU2710700A (en) | 2000-07-03 |
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