Targeting ultrashort fragments: Development and validation of a 44-InDel panel for highly degraded DNA

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• Degraded DNA samples pose significant challenges in forensic casework, often yielding incomplete genetic profiles.
• Existing methods struggle to obtain complete DNA profiles from single-source, highly degraded samples.

Purpose of the Study:

• To develop and evaluate an effective method for accurate genotyping of highly degraded DNA samples.
• To establish a multiplex detection system utilizing Insertion/deletion (InDel) genetic markers for forensic analysis.

Main Methods:

• Selected 44 InDel genetic markers with short amplicon sizes (≤125 bp) and an amelogenin locus.
• Developed a five-dye multiplex PCR system for simultaneous detection of these markers.
• Evaluated the system's performance based on SWGDAM guidelines, including sensitivity, inhibitor tolerance, and degraded sample analysis.

Main Results:

• Achieved complete and reliable DNA profiles with as little as 62.5 pg of input DNA.
• The 44-InDel panel successfully generated complete profiles from DNA subjected to 60 minutes of boiling, outperforming STR-based methods.
• Population analysis in Southern Han Chinese demonstrated high discriminatory power with a combined random match probability of 2.9522 × 10⁻¹⁹.

Conclusions:

• The 44-InDel multiplex system is highly effective for the individual identification of highly degraded forensic samples.
• This InDel panel serves as a valuable complementary tool for forensic casework and paternity testing.
• The short amplicon sizes and robustness of InDels make them ideal for analyzing challenging degraded DNA evidence.