METHOD AND KIT FOR DETERMINING THE GENOME INTEGRITY AND/OR THE QUALITY OF A LIBRARY OF DNA SEQUENCES OBTAINED BY DETERMINISTIC RESTRICTION SITE WHOLE GENOME AMPLIFICATION

• US 20190316194A1
• Filed: 06/27/2019
• Published: 10/17/2019
• Est. Priority Date: 12/04/2013
• Status: Abandoned Application

• Current Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V., Menarini Silicon Biosystems S.P.A
• Original Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V., Menarini Silicon Biosystems S.P.A
• Inventors: Klein, Christoph Andreas, Polzer, Bernhard Michael, Manaresi, Nicolo
• Application Number:
  US16/455,470
• Publication Number:
  US 20190316194A1
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C40B 30/00 Methods of screening libraries
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

A method for determining the integrity of the genome of a sample and/or the quality of a library of DNA sequences obtained by deterministic restriction site whole genome amplification can include (a) amplifying the library of DNA sequences to produce first, second, and third PCR products each of a different size from 50 bp to 1000 bp, by PCR using at least one first primer pair, one second primer pair and one third primer pair, the primer pairs each hybridizing to a DNA sequence of the library having a length from 1000 bp to 5000 bp and corresponding to a sequence of the genome located respectively on a first, second and third chromosome arm; (b) detecting the first, second and third PCR products; (c) correlating the presence of the first, second and third PCR products with the integrity of the genome of the sample and/or the quality of the library.

Claims

1-15. -15. (canceled)

16. A kit for determining the integrity of the genome of a sample and/or the quality of a library of DNA sequences obtained by deterministic restriction site whole genome amplification (DRS-WGA) of the genome of the sample, the kit comprising:

at least one first primer pair which hybridizes to a DNA sequence of the library having a length from 1000 bp to 5000 bp and encompasses the D5S2117 region of chromosome 5q,

at least one second primer pair which hybridizes to a DNA sequence of the library having a length from 1000 bp to 5000 bp and encompasses exons 2 and 3 of the TRP53 gene of the genome, and

at least one third primer pair which hybridizes to a DNA sequence of the library having a length from 1000 bp to 5000 bp and encompasses the KRT19psuedo-gene 1 of the genome,

wherein the first, second and third primer pairs give rise, when amplified by PCR, to PCR products having different size from one another.

17. The kit of claim 16, wherein when amplified by PCR, the first second, and third primer pairs give rise to a first PCR product from 50 bp to 1000 bp, a second PCR product from 50 bp to 1000 bp having a size other than the first PCR product, and a third PCR product from 50 bp to 1000 bp having a size other than the first and second PCR products.

18. The kit of claim 16, wherein a forward primer of the at least one first primer pair is SEQ ID NO:

4 and a reverse primer of the at least one first primer pair is SEQ ID NO;

3.

19. The kit of claim 16, wherein a forward primer of the at least one second primer pair is SEQ ID NO:

6 and a reverse primer of the at least one second primer pair is SEQ ID NO;

5.

20. The kit of claim 16, wherein a forward primer of the at least one third primer pair is SEQ ID NO:

8 and a reverse primer of the at least one third primer pair is SEQ ID NO;

7.

21. The kit of claim 16, further comprising instructions to amplify the library of DNA sequences by PCR using the at least one first primer pair, the at least one second primer pair, and the at least one third primer pair, and to detect the first, second, and third PCR products, wherein the presence of any one or more of the first, second, and third PCR products corresponds to a level of the integrity of the genome of the sample and/or the quality of the library of DNA sequences.

22. The kit of claim 16, further comprising at least one fourth primer pair which hybridizes to a DNA sequence of the library having a length from 80 bp to 300 bp, wherein, when amplified by PCR, a fourth PCR product results having a size other than the first, second, and third PCR product.

23. The kit of claim 22, wherein the fourth PCR product is from 50 bp to 200 bp.

24. The kit of claim 22, wherein the DNA sequence of the library to which the at least one fourth primer pair hybridises encompasses Codon 12/13 of the KRAS gene.

25. The kit of claim 23, wherein a forward primer of the at least one fourth primer pair is SEQ ID NO:

17 and a reverse primer of the at least one first primer pair is SEQ ID NO;

18.

26. The kit of claim 22, further comprising instructions to amplify the library of DNA sequences by PCR using the at least one first primer pair, the at least one second primer pair, the at least one third primer pair, and the at lea tone fourth primer pair and to detect the first, second, third, and fourth PCR products, wherein the presence of any one or more of the first, second, third, and fourth PCR products corresponds to a level of the integrity of the genome of the sample and/or the quality of the library of DNA sequences.

27. The kit of claim 16, wherein the sample consists of 5 cells or less.

28. The kit of claim 16, wherein the sample is a single cell.

29. A method of predicting the success of a genetic analysis assay after amplification of single cell genomic DNA by DRS-WGA comprising amplifying a library of DNA sequences by PCR using the kit of claim 16 and detecting the first, second, and third PCR products.

30. The method of claim 29, wherein the genetic analysis assay is selected from the group consisting of Sanger sequencing for mutation analysis, assessment of specific copy number chambers, quantitative PCR for gene amplification, metaphase Comparative Genomic Hybridization (CGH), and array Comparative Genomic Hybridization (CGH).

31. A method of determining the integrity of the genome of a sample, comprising amplifying a library of DNA sequences by PCR using the kit of claim 16 and detecting the first, second, and third PCR products.

32. The method of claim 31, wherein determining the integrity of the genome of a sample allows to determine a biological status of the cell/cells of the sample.