1. AP Biology Polymerase Chain Reaction March 18, 2014

2. Invented by Kary Mullis Mullis and Faloona, 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction Nobel Prize 1993

3. PCR Targets and amplifies a S SS SINGLE sequence from within a complex mixture of DNA Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA Primary materials, or reagents, used in PCR are: - “Free” DNA nucleotides: building blocks for new DNA - Template DNA: the DNA sequence you want to amplify - Primers: single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA - DNA polymerase: a heat stable enzyme that catalyzes the synthesis of new DNA

4. Heat-stable polymerase is vital to the process… Thermus aquaticus: The Thermus aquaticus DNA polymerase  Taq  Not permanently destroyed at 94ºC  Optimal temperature is 72ºC

5. Primers We must have some information about the sequence n nn next to the target (or gene) Primers provide specificity –T–They are complementary to opposite strands with 3’ ends pointing towards each other –S–Should have similar melting temperatures –M–Must be in vast excess

6. 3 major steps in PCR; repeated for 20 to 40 cycles. This is done in an automated Thermo Cycler, which can heat and cool reaction tubes in a very short time. Denaturation at around 94°CDenaturation at around 94°C : –During denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle). Annealing at around 54°CAnnealing at around 54°C : –Hydrogen bonds are constantly formed and broken between the single stranded primer and single stranded template. If primers exactly fit template, the hydrogen bonds are so strong that the primer stays attached Extension at around 72°CExtension at around 72°C : –Bases (complementary to template) are coupled to primer on 3' side (polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template) Thermocycling PCR Video

7. PCR Summary

8. Templates for PCR Dried blood Semen stains Vaginal swabs Single hair Fingernail scrapings Insects in Amber Egyptian mummies Buccal Swab Toothbrushes

9. Other applications of PCR Detection of mutations –screen for inherited disorders Detection of HIV –Not standard test given Detect tuberculosis without culturing Prenatal sex determination Preimplantation diagnosis of genetic diseases Forensics Paternity testing

10. Variable Number Tandem Repeat Variable Number Tandem Repeat (or VNTR): location in genome where a short nucleotide sequence is organized as a tandem repeat EX: Mississippi, Missississippi, Mississippippi Found on many chromosomes, and often show variations in length between individuals Each variant acts as an inherited allele, allowing it to be used for personal or parental identification (One VNTR is inherited from each parent) Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting

11. DNA profiles vary from person to person When profiles from a single VNTR locus from unrelated individuals are compared, the profiles are normally different. However it is possible for two individuals to have the same profile at one or two loci by chance. But the chance of more than one person having the same DNA profile at 4, 5, or 6 different VNTR loci is extremely small. When DNA profiles are used for forensic purposes, 4-6 different VNTR loci are analyzed. Variations of VNTR (D1S80) allele lengths in 6 individuals The PCR Song

12. Use of VNTR’s in genetic analysis CODIS database –Combined DNA Index System –13 Highly polymorphic loci have been selected by FBI –Probability 1 in 5.7 X 10 15 When removed from surrounding DNA by PCR methods, their size is determined by gel electrophoresis, and they produce a pattern of bands unique to each individual. When tested with a group of independent VNTR markers, the likelihood of two unrelated individuals having the same allelic pattern is extremely improbable.

13. This segment of DNA is cut at restriction sites 1 and 2, which creates restriction fragments A, B, and C. Which of the following electrophoretic gels represents the separation of these fragments? –Answer a –Answer b –Answer c

14. Comprehension Check: Another way to look at VNTR’s Suspect DNA: ILOVEXXXBIO IXLOVEBIO Suspect DNA: ILOVEXBIO IXXXXXLOVEBIO Suspect DNA: IXXXXXLOVEBIO ILOVEXXXXXBIO Suspect DNA: IXLOVEBIO ILOVEXBIO Suspect DNA: ILOVEXXXBIO IXXXXXLOVEBIO Suspect DNA: ILOVEXXXBIO IXXXLOVEBIO Match the sequences below to the electrophoretic patterns (each “sentence” pair represents homologous chromosomes) Individual 1 Individual 5 Individual 6 Individual 2 Individual 3 Individual 4

15. Problems with PCR Contamination Takes one mismatch early on to amplify the wrong fragment