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Optimization of Human mt DNA Control Region Sequencing for Forensic Applications
Author(s) -
Bourdon Véronique,
Ng Carolyn,
Harris Jessica,
Prinz Mechthild,
Shapiro Eli
Publication year - 2014
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/1556-4029.12426
Subject(s) - hypervariable region , sanger sequencing , mitochondrial dna , dna sequencing , biology , amplicon , computational biology , dna , polymerase chain reaction , genetics , microbiology and biotechnology , gene
Sequencing mitochondrial DNA hypervariable regions I and II ( HVI and HVII ) is useful in forensic missing person and unidentified remains cases. Improvements in ease and sensitivity of testing will yield results from more samples in a timely fashion. Routinely, amplification of HVI and HVII is followed by Sanger sequencing using the BigDye ® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) using 4 μL of ready reaction mix ( RRM ). Each sequencing reaction is then purified through column filtration before capillary electrophoresis. Using lower amounts of RRM (2 μL or 1 μL) and purification using BigDye ® XT erminator ™ (Applied Biosystems) instead of columns showed no loss of sequence length and increased the quality and the sensitivity of testing, allowing HVI and HVII typing from mitochondrial genome equivalent to 125 fg of nuclear DNA , or 100 pg of HVI / HVII amplicons. Using this methodology, testing can be completed in 1 day, and the cost of testing is reduced.