AP BiologyPolymerase Chain Reaction

March 18, 2014

Invented by Kary Mullis

• Mullis and Faloona, 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction

• Nobel Prize 1993

PCR• Targets and amplifies a SINGLESINGLE 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

Heat-stable polymerase is vitalto the process…

Thermus aquaticus:

The Thermus aquaticus DNA polymeraseTaqNot permanently destroyed at 94ºCOptimal temperature is 72ºC

Primers• We must have some information about the

sequence next to the target next to the target (or gene)

• Primers provide specificity

– They are complementary to opposite strands with 3’ ends pointing towards each other

– Should have similar melting temperatures

– Must be in vast excess

• 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°C Denaturation 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°C Annealing 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°C Extension 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

PCR Summary

Templates for PCR

• Dried blood• Semen stains• Vaginal swabs• Single hair• Fingernail scrapings• Insects in Amber• Egyptian mummies• Buccal Swab• Toothbrushes

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

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

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

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 1015

• 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.

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

Comprehension Check: Another way to look at

VNTR’s

Suspect DNA: ILOVEXXXBIO IXLOVEBIOSuspect DNA: ILOVEXBIO IXXXXXLOVEBIOSuspect DNA: IXXXXXLOVEBIO ILOVEXXXXXBIOSuspect DNA: IXLOVEBIO ILOVEXBIOSuspect DNA: ILOVEXXXBIO IXXXXXLOVEBIOSuspect 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

Problems with PCR

• Contamination

• Takes one mismatch early on to amplify the wrong fragment