1. Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler June 2009 Chapter 7 DNA Amplification

2. Chapter 7 – DNA Amplification Chapter Summary Detection of minute amounts of biological material from crime scene evidence is possible due to DNA amplification via the polymerase chain reaction (PCR). Using multiplex PCR, specific regions of the human genome are simultaneously targeted with sequence-specific oligonucleotide PCR primers and copied with a DNA polymerase and deoxynucleotide triphosphate building blocks. Close to a billion copies of each specific region of the genome can be generated in a matter of a few hours by subjecting the DNA sample to typically 28- 32 cycles of heating and cooling that permit the replication process to occur. The PCR amplification process incorporates fluorescently- labeled primers into the PCR products that enable multi-color fluorescence detection. In order to protect from contamination by post-PCR products, pre- and post-PCR steps are typically segregated in different laboratory space.

3. PCR Amplification PCR = polymerase chain reaction Process that copies a particular region of DNA using two “primers” (short pieces of DNA) Each strand of DNA is used as a template to create a replicate that permits a doubling of the number of target molecules with each cycle of heating and cooling

4. PCR Process Separate strands (denature) Repeat Cycle, Copying DNA Exponentially Make copies (extend primers) 5’ 3’ Starting DNA Template 80-500 bases Add primers (anneal) 5’ 3’ 5’ Forward Primer Reverse Primer

5. Separate strands (denature) Add primers (anneal) Make copies (extend primers) Repeat Cycle, Copying DNA Exponentially Starting DNA Template 5’ 3’ 5’ 3’ 5’ Forward primer Reverse primer John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 7.2

6. PCR Reaction Setup DNA sample is added (about 1 ng based on DNA quantitation performed) – 10 µL PCR primers and other reaction chemicals from an STR typing kit are added – 15 µL Strip of 8 tubes containing ~25 µL of solution

7. PCR Amplification (Thermal Cycling) The polymerase chain reaction (PCR) copies sections of DNA through heating and cooling the sample Each DNA strand is copied with each temperature cycle A thermal cycler heats and cools DNA samples (usually 28 cycles) ABI 9700 Thermal Cycler

8. 94 o C 60 o C 72 o C Time Temperature Single Cycle Typically 25-35 cycles performed during PCR 94 o C 60 o C 72 o C The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 7.1 PCR Thermal Cycling Temperature Profile

9. John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 7.3 GeneAmp 9700 Thermal Cycler

10. (a) (b) John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 7.4 a set of 8 PCR strip-tubes with attached caps 96-well plate with foil cover

11. (a) Simultaneous amplification of three locations on a DNA template Locus A Locus C Locus B (b) Resolution of PCR products with size-based separation method A C B small large John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 7.5 Multiplex PCR Schematic of Multiple Targets

12. Chapter 7 – Points for Discussion What challenges exist with designing multiplex PCR primers? What are the advantages of a thermal stable, hot-start DNA polymerase? Why is it important to separate pre-PCR and post-PCR processes? What is the purpose of a negative amplification control? A postive amplification control? What are some effective means to prevent contamination? What are some effective means to clean up following a contamination event? What are “stochastic effects” and why are they important to forensic DNA analysis?