Development of a highly sensitive quantification system for assessing DNA degradation and quality in forensic samples

• US 9,957,557 B2
• Filed: 08/12/2013
• Issued: 05/01/2018
• Est. Priority Date: 08/13/2012
• Status: Active Grant

• Current Assignee: Life Genetics Lab, LLC
• Original Assignee: Life Genetics Lab, LLC
• Inventors: Sinha, Sudhir
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/964,970
• Publication Number:
  US 20140051075A1
• Time in Patent Office: 1,723 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 435183, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

A process of quantifying the extent of degradation present in a human DNA sample is described. The process makes use of a real time PCR system to separately quantitate within a sample a first retrotransposon interspersed element and a relatively longer second retrotransposon interspersed element, where the longer element is expected to be disrupted at a faster pace than is the shorter element as the sample degrades. In one embodiment, the process makes use of the appearance of the relatively young (on an evolutionary scale) Alu Yb-lineage subfamily sequences appearing in every human genome and their virtual absence in non-human samples. In a preferred embodiment, the process quantifies longer 290 bp sequences of “SVA” elements and shorter 80 bp sequences of Alu Yb8-lineage. Newly designed primers and TaqMan probes that are useful in the process are presented. A related process additionally quantifies male specific human DNA.

Claims

1. A process for assessing the extent of degradation of human DNA in a sample, the process comprising the steps of:

providing a sample containing human DNA;

quantitating an amount of a first retrotransposon interspersed element in the sample using a real-time polymerase chain reaction (PCR) technique, and quantitating an amount of a second retrotransposon interspersed element in the sample using a real-time polymerase chain reaction technique, the first retrotransposon interspersed element being an Alu element found in the human genome, the second retrotransposon interspersed element being an SVA element of the human retinitis pigmentosa (RP) gene;

determining a ratio of the amount of the first retrotransposon interspersed element to the amount of the second retrotransposon interspersed element in the sample; and

assessing the extent of degradation of human DNA in the sample based on said ratio.

2. The process according to claim 1, said quantitating the amount of the first retrotransposon interspersed element and said quantitating the amount of the second retrotransposon interspersed element being performed simultaneously.

3. The process of claim 2, the real time polymerase chain reaction technique is operated under the following conditions:

95°

C. for 10 minutes;

32-40 cycles of;

95°

C. for 15 seconds, and 61°

C. for 70-120 seconds.

4. The process of claim 1, the second retrotransposon interspersed element comprising at least three times as many base pairs as the first retrotransposon interspersed element comprises.

5. The process of claim 1, the first retrotransposon interspersed element consists of 40 to 150 base pairs, the second retrotransposon interspersed element consists of 120 to 400 base pairs.

6. The process of claim 1, the process further comprises the preparation of standard curves for the quantitation of the first retrotransposon interspersed element and the quantitation of the second retrotransposon interspersed element.

7. The process of claim 1, the real time polymerase chain reaction technique does not amplify non-primate DNA.

8. The process of claim 1, the sample comprising DNA which has been degraded by mechanical means.

9. The process of claim 1, the sample comprising DNA which has been degraded by chemical means.

10. The process of claim 1, the sample comprising DNA which has been degraded by environmental means.

11. The process of claim 1, where the Alu element is an Alu Yb8 element.

12. A process for assessing the extent of degradation of human DNA in a sample, the process comprising the steps of:

providing a sample containing human DNA;

providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO;

5 and a reverse primer labeled SEQ ID NO;

6; and

providing at least one primer selected from the group consisting of a forward primer labeled SEQ ID NO;

8, a forward primer labeled SEQ ID NO;

11, a forward primer labeled SEQ ID NO;

14, a reverse primer labeled SEQ ID NO;

9, a reverse primer labeled SEQ ID NO;

12, a reverse primer labeled SEQ ID NO;

13 and a reverse primer labeled SEQ ID NO;

15;