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 20180187254A1
• Filed: 12/04/2014
• Published: 07/05/2018
• Est. Priority Date: 12/04/2013
• Status: Active Grant

• 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
• Granted Patent: US 10,392,657 B2
• 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. 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 (DRS-WGA) of the genome of the sample comprising the steps of:

(a) amplifying the library of DNA sequences by PCR using;

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 corresponding to a sequence of the genome located on a first chromosome arm,

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 corresponding to a sequence of the genome located on a second chromosome arm other than the first chromosome arm, 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 corresponding to a sequence of the genome located on a third chromosome arm other than the first and second chromosome arms,

wherein the step of amplifying giving 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;

(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 of DNA sequences.

2. The method according to claim 1, 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.

3. The method according to claim 1, 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.

4. The method according to claim 1, 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.

5. The method according to claim 2, wherein step (a) further uses at least one fourth primer pair which hybridizes to a DNA sequence of the library having a length from 80 bp to 300 bp, the step of amplifying giving rise to a fourth PCR product from 50 bp to 200 bp having a size other than the first, the second and the third PCR products;

step (b) further comprises detecting the fourth PCR product;

step (c) further comprises correlating the presence of the fourth PCR product with the integrity of the genome of the sample and/or the quality of the library of DNA sequences.

6. The method according to claim 5, 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.

7. The method according to claim 6, 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.

8. 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 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 corresponding to a sequence of the genome located on a first chromosome arm,

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 corresponding to a sequence of the genome located on a second chromosome arm other than the first chromosome arm, 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 corresponding to a sequence of the genome located on a third chromosome arm other than the first and second chromosome arms,

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

9. The kit according to claim 8, 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 the first, second, third and fourth primer pairs give rise, when amplified by PCR, to PCR products having different size from one another.

10. Use of the kit according to claim 8 in predicting the success of a genetic analysis assay after amplification of single cell genomic DNA by DRS-WGA.

11. The use according to claim 10, wherein the genetic analysis assay is selected from the group consisting of Sanger sequencing for mutation analysis, assessment of specific copy number changes, quantitative PCR for gene amplification, metaphase Comparative Genomic Hybridisation (CGH), and array Comparative Genomic Hybridisation (CGH).

12. The kit according to claim 8 for use in determining the integrity of the genome of a sample.

13. The kit according to claim 12, wherein the sample consists of 5 cells or less.

14. The kit according to claim 13, wherein the sample is a single cell.

15. The kit according to claim 13, wherein determining the integrity of the genome of the sample allows to determine the biological status of the cell/cells of the sample.