Modified midi- and mini-multiplex PCR systems for mitochondrial DNA control region sequence analysis in degraded...

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Mitochondrial DNA (mtDNA) analysis is crucial for identifying degraded or ancient samples.
• Traditional monoplex PCR methods can be inefficient with limited or degraded DNA.
• Developing robust amplification strategies for challenging samples is essential.

Purpose of the Study:

• To develop and validate multiplex polymerase chain reaction (PCR) systems for amplifying the mitochondrial DNA (mtDNA) control region.
• To assess the efficiency of these multiplex systems in amplifying degraded DNA from ancient skeletal remains.
• To provide a more effective and economical approach for mtDNA sequencing in forensic and ancient DNA studies.

Main Methods:

• Development of two multiplex PCR systems: Midiplex (two multiplexes, six amplicons) and Miniplex (three multiplexes, ten amplicons).
• Amplification of overlapping amplicons ranging from 142 to 267 base pairs (bp).
• Validation using old skeletal remains (60-year-old and 400-500-year-old) to test degraded DNA amplification efficiency.

Main Results:

• Successful amplification and sequencing of mtDNA control region from ancient skeletal remains using both Midiplex and Miniplex systems.
• Obtained sequences were consistent with those from previous monoplex amplification methods.
• Demonstrated high efficiency in amplifying degraded DNA samples.

Conclusions:

• Multiplex PCR systems, particularly those with smaller amplicons (Miniplex), are effective for mtDNA sequence analysis of ancient and degraded forensic samples.
• These optimized multiplex systems significantly save time, cost, and DNA sample consumption.
• The developed systems represent a valuable advancement for forensic and ancient DNA research.