Methods for rapid forensic analysis of mitochondrial DNA

a mitochondrial dna and forensic analysis technology, applied in the field of mitochondrial dna analysis, can solve the problems of inability to detect low levels of heteroplasmy in detection methods currently available to molecular biologists, difficult recording and comparing mtdna sequences, and potentially confusing,

Inactive Publication Date: 2005-12-01
IBIS BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recording and comparing mtDNA sequences would be difficult and potentially confusing if all of the bases were listed.
Presently, the forensic analysis of mtDNA is rigorous and labor-intensive.
Heteroplasmy is a problem for forensic investigators since a sample from a crime scene can differ from a sample from a suspect by one base pair and this difference may be interpreted as sufficient evidence to eliminate that individual as the suspect.
The detection methods currently available to molecular biologists cannot detect low levels of heteroplasmy.
Furthermore, if present, length heteroplasmy will adversely affect sequencing runs by resulting in an out-of-frame sequence that cannot be interpreted.
However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.

Method used

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  • Methods for rapid forensic analysis of mitochondrial DNA
  • Methods for rapid forensic analysis of mitochondrial DNA
  • Methods for rapid forensic analysis of mitochondrial DNA

Examples

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example 1

Nucleic Acid Isolation and Amplification

[0075] General Genomic DNA Sample Prep Protocol: Raw samples were filtered using Supor-200 0.2 μm membrane syringe filters (VWR International). Samples were transferred to 1.5 ml eppendorf tubes pre-filled with 0.45 g of 0.7 mm Zirconia beads followed by the addition of 350 μl of ATL buffer (Qiagen, Valencia, Calif.). The samples were subjected to bead beating for 10 minutes at a frequency of 19 l / s in a Retsch Vibration Mill (Retsch). After centrifugation, samples were transferred to an S-block plate (Qiagen, Valencia, Calif.) and DNA isolation was completed with a BioRobot 8000 nucleic acid isolation robot (Qiagen, Valencia, Calif.).

[0076] Isolation of Blood DNA—Blood DNA was isolated using an MDx Biorobot according to according to the manufacturer's recommended procedure (Isolation of blood DNA on Qiagen QIAamp® DNA Blood BioRobot® MDx Kit, Qiagen, Valencia, Calif.)

[0077] Isolation of Buccal Swab DNA—Since the manufacturer does not suppo...

example 2

Digestion of Amplicons with Restriction Enzymes

[0080] Reaction Conditions—The standard restriction digest reaction conditions outlined herein are applicable to all panels of restriction enzymes. The PCR reaction mixture is diluted into 2×NEB buffer 1+BSA and 1 μl of each enzyme per 50 μl of reaction mixture is added. The mixture is incubated at 37° C. for 1 hour followed by 72° C. for 15 minutes. Restriction digest enzyme panels for HV1, HV2 and twelve additional regions of mitochondrial DNA are indicated in Table 2.

TABLE 2mtDNA Regions, Coordinates and Restriction Enzyme Digest PanelsCOORDINATES RELATIVETO THE ANDERSONRESTRICTIONmtDNA REGIONSEQUENCE (SEQ ID NO: 72)ENZYME PANELHV1 (highly variable16050-16410RsaIcontrol region 1)HV2 (highly variable 29-429HaeIII HpaII MfeI SspIcontrol region 2)orHpaII, HpyCH4IV, PacI andEaeIREGION R1 (COX2,8162-8992DdeI MseI HaeIII MboIIntergenic spacer, tRNA-LYS, ATP6)REGION R2 (ND5)12438-13189DdeI HaeIII MboI MseIREGION R3 (ND6 tRNA-Glu,14629-15...

example 3

Nucleic Acid Purification

[0081] Procedure for Semi-Automated Purification of a PCR Mixture Using Commercially Available Zip Tips®—As Described by Jiang and Hofstadler (Y. Jiang and S. A. Hofstadler Anal. Biochem. 2003, 316, 50-57) an amplified nucleic acid mixture can be purified by commercially available pipette tips containing anion exchange resin. For pre-treatment of ZipTips® AX (Millipore Corp. Bedford, Mass.), the following steps were programmed to be performed by an Evolution™ P3 liquid handler (Perkin Elmer) with fluids being drawn from stock solutions in individual wells of a 96-well plate (Marshall Bioscience): loading of a rack of ZipTips®AX; washing of ZipTips®AX with 15 μl of 10% NH4OH / 50% methanol; washing of ZipTips® AX with 15 μl of water 8 times; washing of ZipTips® AX with 15 μl of 100 mM NH4OAc.

[0082] For purification of a PCR mixture, 20 μl of crude PCR product was transferred to individual wells of a MJ Research plate using a BioHit (Helsinki, Finland) multich...

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Abstract

The present invention provides methods for rapid forensic analysis of mitochondrial DNA by amplification of a segment of mitochondrial DNA containing restriction sites, digesting the mitochondrial DNA segments with restriction enzymes, determining the molecular masses of the restriction fragments and comparing the molecular masses with the molecular masses of theoretical restriction digests of known mitochondrial DNA sequences stored in a database.

Description

FIELD OF THE INVENTION [0001] This invention relates to the field of mitochondrial DNA analysis. The invention enables rapid and accurate forensic analysis by using mass spectrometry to characterize informative regions of mitochondrial DNA. BACKGROUND OF THE INVENTION [0002] Mitochondrial DNA (mtDNA) is found in eukaryotes and differs from nuclear DNA in its location, its sequence, its quantity in the cell, and its mode of inheritance. The nucleus of the cell contains two sets of 23 chromosomes, one paternal set and one maternal set. However, cells may contain hundreds to thousands of mitochondria, each of which may contain several copies of mtDNA. Nuclear DNA has many more bases than mtDNA, but mtDNA is present in many more copies than nuclear DNA. This characteristic of mtDNA is useful in situations where the amount of DNA in a sample is very limited. Typical sources of DNA recovered from crime scenes include hair, bones, teeth, and body fluids such as saliva, semen, and blood. [0...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N27/447G01N33/48G01N33/50G06F19/00G16B50/30
CPCC12Q1/683C12Q1/6846G01N27/447G06F19/28G01N27/62C12Q2565/627C12Q2521/301G16B50/00G16B50/30G01N27/623C12Q1/68
Inventor HOFSTADLER, STEVENHALL, THOMASECKER, DAVIDBLYN, LAWRENCEESHOO, MARKSAMANT, VIVEKWHITE, NEILL
Owner IBIS BIOSCI
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