FSCT 8150 Week 4 Notes: DNA Amplification, PCR, and STR Kits

DNA Amplification and PCR

• Chapter 7 covers DNA amplification and the PCR process in detail.

• Read the textbook chapter to gain a foundational understanding of PCR before watching the video lectures.

• Delve into the provided links to fully grasp the principles of what PCR is and how it functions at a molecular level.

• Place a significant emphasis on the commercial kits available from leading suppliers such as Applied Biosystems (Thermo Fisher), Promega, and QIAGEN, understanding their specific applications and advantages.

PCR Process Overview

• Each PCR cycle effectively doubles the quantity of template DNA present, leading to exponential amplification.

• Starting with a single DNA template molecule, each cycle results in a doubling of the DNA, initiating the exponential amplification process.

• The cyclical repetition of PCR steps results in exponential amplification, where the DNA quantity increases exponentially (e.g., $1 \rightarrow 2 \rightarrow 4 \rightarrow 8$\rightarrow and so on).

• The PCR process consists of three primary steps:

  • Denaturation: The double-stranded DNA template is heated to a high temperature (typically $94-96$ degrees Celsius) in the thermocycler to disrupt the hydrogen bonds and separate it into single strands. This step is crucial for primer binding and subsequent DNA synthesis.

  • Annealing: The temperature is lowered to allow primers to bind to their complementary sequences on the single-stranded DNA template. The annealing temperature is optimized based on the primer sequence, typically ranging from $50$ to $65$ degrees Celsius.

  • Extension: The DNA polymerase enzyme extends the primers by adding nucleotides to synthesize a new DNA strand complementary to the template strand. The extension temperature is optimal for the polymerase enzyme, typically around $72$ degrees Celsius.

Cycling Parameters and Optimization

• During denaturation, double-stranded DNA is heated to $94$ degrees Celsius to ensure complete separation into single strands, which is critical for subsequent primer annealing.

• The temperature is then decreased to $60$ degrees Celsius to facilitate the binding of primers to their complementary sequences on the single-stranded DNA.

• In the extension phase, the temperature is raised to $72$ degrees Celsius to enable optimal activity of the DNA polymerase enzyme to synthesize new DNA strands.

• Standard PCR protocols typically involve 28, 29, or 30 cycles to achieve sufficient DNA amplification for downstream analysis.

Global Filer Thermocycline Profile

• The Global Filer thermocycline profile is optimized to minimize the overall PCR run time while maintaining amplification efficiency.

• Initial incubation at $95$ degrees Celsius for one minute is performed to activate the DNA polymerase enzyme, ensuring it is ready for subsequent DNA synthesis.

• The denaturation phase is carried out at $94$ degrees Celsius for ten seconds to separate the DNA strands rapidly.

• Annealing and extension occur simultaneously at $59$ degrees Celsius for ninety seconds, streamlining the PCR process.

• A final extension step is performed for ten minutes to ensure complete extension of all DNA fragments, which is reduced from the traditional 30-40 minutes.

• The samples are then held at a holding temperature of $4$ degrees Celsius until further analysis.

Evolution of PCR Amplification Kits (Promega Example)

• Over the years, there has been a notable trend toward incorporating a greater number of STR markers in PCR amplification kits to enhance forensic DNA analysis capabilities.

• Buffers have undergone modification to enhance their effectiveness, particularly in the presence of PCR inhibitors commonly found in forensic samples.

• Primer designs have been optimized to amplify shorter STR regions by moving primers closer, resulting in improved amplification of degraded DNA samples, which is crucial in forensic investigations.

PCR Amplification Kits

Profiler Plus Kit

• The Profiler Plus kit, introduced in 1997 by Applied Biosystems, marked a significant advancement in PCR-based DNA analysis.

• It was widely adopted by forensic DNA laboratories in Canada and the United States, including the RCMP and the Centre of Forensic Science in Ontario, as well as being utilized for educational purposes at BCIT.

• The kit targeted nine STR loci of the CODIS loci, along with amelogenin, a gender-determining locus, providing valuable information for forensic identification purposes.

  • CODIS: Combined DNA Index System is the FBI's comprehensive database containing DNA profiles for forensic and criminal justice applications.

• The Profiler Plus kit employed four fluorescent dyes to enable multiplex PCR amplification and detection of the targeted STR loci.

• A notable limitation of the kit was its susceptibility to degradation due to the larger fragment sizes (up to 405 base pairs), which could compromise the analysis of degraded DNA samples.

Analyzing a Profile

• Zooming in on a profile is essential for assessing baseline noise levels and identifying potential artifacts that may affect data interpretation.

• Conversely, zooming out provides a comprehensive view of all alleles present in the profile, aiding in overall assessment and comparison. Example: Profiler Plus profile with blue, green, and yellow channels for visualizing different STR loci.

• The amelogenin location on the profile serves as an indicator of gender, with XX indicating a female and XY indicating a male.

• Stutter peaks, which are artifacts of the PCR process, must be filtered out to ensure accurate and reliable allele calling and data interpretation.

Cofiler Kit

• The Cofiler kit, also developed by Applied Biosystems, was utilized concurrently with the Profiler Plus kit in forensic DNA analysis.

• It served as an add-on kit to supplement the Profiler Plus when additional markers were required or when the DNA profile was degraded.

• Some markers overlapped with Profiler Plus, such as D3 and D7, providing valuable quality control measures to ensure data consistency and accuracy.

Identifiler Kit

• The Identifiler kit, released in 2001 by Applied Biosystems, represented a significant advancement in STR analysis, incorporating an expanded set of markers for enhanced discrimination.

• It included 13 CODIS loci, along with D2 and D19, bringing the total to 15 STR loci, in addition to amelogenin for gender determination.

• This kit was the first to incorporate mobility modifying non-nucleotide linkers to adjust marker size and prevent overlap, enabling the accommodation of more markers in a single multiplex PCR assay.

• The Identifiler kit utilized five-dye fluorescent technology, providing increased multiplexing capability and improved data resolution.

• The size range of amplified fragments spanned from 100 to 360 base pairs, optimizing amplification efficiency and data quality.

Allelic Ladder

• The allelic ladder comprises all commonly found alleles in the population at each locus, serving as a reference standard for accurate allele calling.

• It is used to set bins for allele calling in the analysis software, ensuring consistency and reliability in data interpretation.

PowerPlex System

• The PowerPlex System, introduced by Promega in February, offered a comprehensive solution for STR analysis with an expanded marker set.

• It included 15 STR loci, including 13 CODIS loci, along with Penta E, Penta D, and amelogenin, providing comprehensive forensic DNA profiling capabilities.

• The kit employed four-dye technology for multiplex PCR amplification and detection of the targeted STR loci.

• Compared to Identifiler, the PowerPlex System exhibited larger fragment sizes, reaching up to 475 base pairs, which could pose challenges when analyzing degraded samples.

Degraded samples

• Larger fragments are more susceptible to degradation, potentially leading to incomplete or inaccurate DNA profiles during analysis.

Mini STRs

Introduction

• Mini STRs were introduced in 2007 with the release of the MiniFiler kit by Applied Biosystems, addressing the challenges posed by degraded or inhibited DNA samples.

• Designed specifically for degraded or inhibited samples, Mini STRs feature primer binding sites located closer to the STR region, resulting in shorter amplicons.

• Mini STR kits utilized non-nucleotide linkers and five-dye technology, enhancing multiplexing capability and data resolution.

• Typically, Mini STR kits target eight loci, providing a focused set of markers for efficient analysis of compromised DNA samples.

• Partial overlap with ProfilePlus was incorporated for quality control purposes, ensuring compatibility and reliability in data comparison.

Case Example: BCIT Forensic DNA Lab (2007)

• In a notable case study from the BCIT Forensic DNA Lab in 2007, the application of Mini STRs proved instrumental in resolving a cold case involving a single-engine plane crash in 1970.

• The discovery of a skull with a gold tooth by a hiker prompted forensic investigation efforts.

• Initial analysis using Profiler Plus yielded limited information due to the degraded nature of the DNA sample.

• However, MiniFiler analysis successfully generated a full DNA profile, enabling kinship analysis and ultimately leading to the identification of the crash victim.

Next Generation Amplification Kits

Developments

• Promega introduced PowerPlex 16 Hot Start in 2009, while Applied Biosystems released the Identifiler Plus kit in 2010, marking advancements in PCR amplification technology.

• These kits featured improved reagents to enhance sensitivity and effectively overcome PCR inhibition, enabling more robust and reliable DNA amplification.

• The incorporation of improved DNA polymerases further enhanced amplification efficiency and fidelity.

Kits in Europe

• Historically, QIAGEN kits were predominantly sold in Europe due to patent restrictions affecting North America and Japan.

• Forensic DNA laboratories in Europe have a long-standing history of utilizing QIAGEN kits for STR analysis.

• Popular kits in Europe included SGM Plus, SEfiler (Applied Biosystems), NGM, and PowerPlex ESI/ESX (Promega), catering to diverse forensic DNA profiling needs.

• Efforts are underway to harmonize DNA testing practices between Europe and North America to facilitate international data sharing and collaboration.

• Applied Biosystems introduced GlobalFiler, while Promega developed PowerPlex Fusion, representing advancements in multiplex PCR technology.

• The FBI recommended the inclusion of additional loci to augment the power of discrimination in forensic DNA analysis, enhancing the accuracy and reliability of identification.

Promega Power Plex Fusion Kit

• The Promega Power Plex Fusion Kit offers an expanded marker set comprising 24 loci, enhancing discriminatory power in forensic DNA analysis.

• It is amenable to both DNA extracts and direct amplification of reference samples such as blood and buccal swabs, providing versatility in sample processing.

Fusion 6C Kit

• The Fusion 6C Kit represents an updated iteration featuring six colors and targeting 27 loci, further expanding the scope of forensic DNA profiling.

• It includes 23 autosomal STRs, three Y STRs, and amelogenin, providing comprehensive genetic information for individual identification.

• The inclusion of YSTR markers offers additional benefits, such as confirming gender or addressing instances of missing amelogenin, enhancing the robustness of the analysis.

GlobalFiler and GlobalFiler Express

• GlobalFiler and GlobalFiler Express represent a new generation of 24-loci multiplex assays released around 2014 or 2015, offering enhanced capabilities in forensic DNA analysis.

• GlobalFiler incorporates ten mini STRs, specifically designed for improved amplification of degraded DNA samples, ensuring robust performance in challenging forensic casework.

• GlobalFiler Express is designed as a direct amplification kit, streamlining the workflow for reference samples and enabling rapid processing.

• Both kits feature the amelogenin marker and an insertion-deletion marker on the Y chromosome for reliable gender determination.

• The presence of multiple markers for gender determination represents an improvement over previous systems, enhancing the accuracy and reliability of gender identification.

Evolution of Applied Biosystems Kits

Progression

• The evolution of Applied Biosystems kits follows a progression from Profiler Plus to Identifiler Plus and ultimately to GlobalFiler, each offering advancements in marker coverage and assay performance.

• The number of loci incorporated within the kits increased from 10 to 16 to 24, enhancing discriminatory power and expanding the scope of forensic DNA analysis.

• The number of autosomal markers also increased within the kits, providing a more comprehensive genetic profile for individual identification.

• The Global Filer kit incorporates three sex markers, improving the accuracy and reliability of gender determination.

• An increased number of mini STRs are included, enabling improved amplification of degraded DNA samples.

• The transition from four to five dyes enhances multiplexing capability and data resolution, improving the efficiency and throughput of forensic DNA analysis.

• The volume of input DNA has increased from 10 to 15, increasing the concentration of the master mix and optimizing amplification efficiency.

Benefits

• These advancements enable reaching optimal DNA input, even at low concentrations, ensuring robust and reliable amplification.

• The amount of time required for PCR amplification is reduced, streamlining the workflow and improving turnaround times.

• Detection platforms have improved over time, leading to increased sensitivity and enhanced sample detection capabilities.

• Overall, these developments lead to the generation of more DNA information in less time, enabling faster turnaround times and more efficient forensic DNA analysis.