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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
  • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/154—Methylation markers

Definitions

• the measurement(s) and analyses can be done independent of purification, storage - and to quite some extent - also to tissue quality.
• the amplification involves a polymerase enzyme, a PCR or chemical amplification reaction, or other amplification methods as known to the person of skill as described below, e.g. in the context of MSP, HeavyMethyl, Scorpion, MS-SNUPE, MethylLight, bisulfite sequencing, methyl specific restriction assays and/or digital PCR (see, for example Kristensen and Hansen PCR-Based Methods for Detecting Single-Locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment Clinical Chemistry 55 :8 1471-1483 (2009)).
• an amplicon of the LRP5 gene region is produced that is a particularly preferred "tool" for performing the method(s) according to the present invention. Consequently, oligomers according to any of SEQ ID No. 4 and 5 or an amplicon as amplified by a primer pair based on SEQ ID No. 4 and 5 or 6 and 7 or 9 and 10 as mentioned herein constitute preferred embodiments of the present invention.
• the sequences of SEQ ID No. 1 to 3 can be used to design primers for amplifications, i.e. serve as "beacons” in the sequence as relevant.
• additional primers and probes can be designed based on the amplicon according to SEQ ID No. 1. Amplification can take place either in the genomic and/or bisulfite (i.e. "converted") DNA sequence.
• the person of skill will furthermore be able to select specific subsets of CpG positions in order to minimize the amount of sites to be analyzed, for example at least one of CpG position selected from a CpG position in an amplicon according to SEQ ID No. 1, and is preferably selected from CpG positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 in the amplicon No. 2249 according to SEQ ID No. 1.
• the sample is selected from a mammalian body fluid, including human blood samples, or a tissue, organ or a sample of leukocytes or a purified or separated fraction of such tissue, organ or leukocytes or a cell type sample.
• a mammal is a mouse, goat, dog, pig, cat, cow rat, monkey or human.
• the samples can be suitably pooled, if required.
• Another preferred aspect of the method according to the present invention then relates to a method as above, further comprising formulating said B cells as identified for transplantation into a patient.
• Pharmaceutical preparations for these purposes and methods for their production are performed according to methods known in the art of transplantation medicine.
• Figure 1 shows the analysis of CpG sites on amplicon No. 2249 (SEQ ID No. 1) according to the invention.
• the horizontal boxes in the table correspond to the CpG positions in the amplicon as analyzed (e.g. CpG 1, 2, etc.) with the positions indicated (105, 114, 159, 163, 167, 171, 174, 194, 208, 213, 218, 223, 261, 272, 285, 306, 336, 357, 384, 423, and 433, corresponding to CpG 3, 4, ...etc.), and the columns correspond to the cell types as analyzed.
• test-templates plasmid-DNA

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• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 2015
  • 2015-09-25 GB GBGB1516972.5A patent/GB201516972D0/en not_active Ceased
• 2016
  • 2016-09-22 HK HK18116376.0A patent/HK1257122A1/zh unknown
  • 2016-09-22 US US15/752,072 patent/US11643687B2/en active Active
  • 2016-09-22 CN CN201680052685.1A patent/CN108026578A/zh active Pending
  • 2016-09-22 JP JP2018515528A patent/JP2018529345A/ja active Pending
  • 2016-09-22 EP EP16770025.1A patent/EP3353318B1/en active Active
  • 2016-09-22 WO PCT/EP2016/072579 patent/WO2017050916A1/en not_active Ceased

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