Introduction to PCR

Kristen Wolslegel Manager, Education Programs

BABEC

What is DNA?

“The single most important molecule in living cells” Molecular Biology, D. Freifelder, 1987 “The prime molecule of life”

Recombinant DNA, Watson, J. et al, 1983

Image from the U. S. Department of Energy Human Genome Project

It encodes within its structure the hereditary information that determines the form and function of all known living organisms.

DNA consists of building blocks called nucleotides

What is DNA made of?

Image: SCFBIO http://www.scfbio-iitd.res.in/tutorial/gene.html

1) a phosphate molecule

gives DNA its negative charge 2) a pentose sugar

five-carbon sugar in ring form 3) a nitrogenous base

ring of carbon and nitrogen atoms variable

2 Types of Nitrogenous Bases (4 in total)

Purines: fused 5 & 6 member rings Adenine & Guanine

Pyrimidines: 6-member ring Cytosine & Thymine

http://www.uic.edu/classes/bios/bios100/lectf03am/lect02.htm

The Base Pairing Rules

  Hydrogen bonds form between bases

A = T two hydrogen bonds

G ≡ C three hydrogen bonds

  The bonds are weak and can be

broken by high temperatures

DNA has two strands with bases paired in the middle

DNA Replication Animation

Molecular Cell Biology, Lodish et. al. 4th ed.

What is the Polymerase Chain Reaction?

A technique in molecular biology used to rapidly amplify a piece of DNA generating

millions of copies of a specific DNA sequence

  The amount of DNA in a cell is too small to be analyzed  It is a method used to generate more DNA than we can extract from cells   It allows for the detection & measurement of DNA from a small sample

Why?

DNA Learning Center Videos

http://www.geneticorigins.org/pv92/aluframeset.htm

http://www.dnalc.org/resources/3d/19-polymerase-chain-reaction.html

Primers

  It’s how we fish out the part of DNA that we’re interested in   The starting point of the reaction   Two short, synthetic DNA segments specific for the region of interest

Primer

DNA Polymerase from Thermus aquaticus!

  A thermostable DNA polymerase Can function at high temperatures Isolated from a thermophilic bacteria that could withstand 95oC

  Discovered in 1965 in the hot springs of Yellowstone National Park   Commonly known as “Taq”

Images: Canadian Agricultural Board http://sci.agr.ca/crda/indust/microscope_e.htm http://culturingscience.wordpress.com/ 2010/06/25/hydrothermal_vent_colonization/

Deoxynucleotide-triphosphates: dNTPs

The “PCR Building Blocks”

A “dNTP mix” contains equal amounts of :

dATP

dTTP

dGTP

dCTP

The PCR “Cycle”

Denature: 94-96oC… Anneal: 37-65oC… Extension: 72oC… Repeat steps 1-3:

Separates double helix into two strands

Primers bind to target site on single stranded DNA

DNA polymerase adds dNTPs according to the base pairing rules (polymerization)

5 to 40 times using a Thermal Cycler

Cycle 1

Cycle 2

Cycle 3

After 30 cycles, DNA is amplified over a billion-fold!

Cycle Relative Amount 1 22 43 84 165 326 647 1288 2569 51210 1,024. .. .20 1,048,576. .. .30 1,073,741,824

Target sequence

Polymerase Chain Reaction Cell

What are we copying?

How do we separate the DNA?

What is doing the copying?

How do we fish out the sequence?

What does the work?

Cellular DNA Replication

PCR vs. Cellular DNA Replication

DNA DNA

Heat Enzymes

Taq polymerase Human polymerase

Primers Primers

Thermal cycler Cell

  Diagnosis of diseases:

  Cancer, microbial & viral infections

  Blood type testing

  Detection of heritable/genetic diseases (amniocentesis)

PCR in Medicine

PCR in the Food Industry

  Detection of microorganisms in drinking water, shellfish, hamburger

  Detection of allergens in food

  Detection of genetically engineered elements in crop plants

PCR in Forensics   Providing evidence from crime scenes: DNA from blood, tissue, hair or bodily fluid

  Exoneration of the wrongly convicted

  Paternity Testing: Thomas Jefferson’s descendants

  Identifying human remains from disasters: World Trade Center

PCR in Anthropology & Evolutionary Biology

  Phylogenic analysis of DNA from ancient sources (Neanderthals, Mammoths)

  Analysis of ancient humans

  Study evolutionary relationships

Image: Penn State http://www.rps.psu.edu/indepth/mammoth/page3.html

Image: Georgia Perimeter College http://higheredbcs.wiley.com/legacy/college/levin /0471697435/chap_tut/chaps/chapter17-05.html

Agarose Gel Electrophoresis

Agarose Gel Electrophoresis

Dye is added to give the DNA color

DNA “Ladder”

well #1

well #2

well #3

+ _

large DNA fragments

small DNA fragments

Electron micrograph of an agarose gel

  DNA sequence that "reproduces" by copying itself and inserting a new copy at a different location in the genome

  Genes comprise only about 5% of chromosomal DNA. The other 95% is non-coding DNA and it’s function is unknown

"  Human chromosomes contain about 1,000,000 Alu copies, which

equal 10% of the total genome."

  Alu PV92 is located on chromosome #16. Alu insertion is stable through evolutionary time.

  If your parents have it, you inherit it from them - not everyone has it. "

Alu is DNA that can move!

Alu may be present or absent on each of your paired chromosomes, creating two possibilities (+ and –)

Inherited from both parents…… Inherited from one parent…….. Not inherited……………………

How is Alu passed on from our parents?

Alu+/Alu- Alu-/Alu-

+/- +/- -/- -/-

+/+ +/- -/-

These are called “alleles”

Alu+/Alu- Alu+/Alu-

+/+ +/- -/+ -/-

715 base pairs

415 base pairs

Student #1

Student #2

Student #3

The Alu PV92 DNA sequence amplified by PCR

PCR Target

Forward primer

Reverse primer chromosome 16 without insert

415 bp (-)

PCR Target

Forward primer

Reverse primer Alu (300 bp)

?? (+)

chromosome 16 with insert

+/+ +/- -/-

  Alu insertions are useful tools for reconstructing human evolution and migration

  Alu PV92 was duplicated within the last several hundred thousand years, reaching different frequencies in different human populations over time

  Who is more related? +/+, +/-, -/-

  We can compare our class data to studies done around the world and map the global spread of the Alu PV92 allele

Alu tells us about human migration

Online bioinformatics exercise

After learning your genotypes, you can calculate the "percent of your class that has the Alu insert"

"Then you can then compare the Alu frequencies of your class"

population to data from other classes around the world"

http://www.bioservers.org/bioserver/

Calculating Allele & Genotype Frequencies

To estimate the frequency of Alu within a population:

1.  Amplify Alu-region from representative

sample population

2. Calculate the observed allelic frequencies and expected genotypic frequencies

Calculating Allele Frequencies

Frequency of (+) = # of (+) alleles # of students

Frequency of (-) = # of (-) alleles # of students

Allele Frequency: the percent of a particular allele within a population

There are twice as many total alleles as there are people

Calculating Allele Frequencies

Homozygous positive: 2 alleles X # (+/+) students = # (+) alleles student Homozygous negative: 2 alleles X # (-/-) students = # (-) alleles student Heterozygous: 1 (+) allele , 1 (-) allele

student student

Calculating Genotype Frequencies

+/+ = p2 +/- = 2pq -/- = q2

pp pq

qq pq

p

p

q

q

The Hardy-Weinberg Equation

p2 + 2pq + q2 = 1

The Hardy-Weinberg Principle

The Hardy-Weinberg principle allows us to calculate the expected genotypes in a population so that we can observe how that"population changes over time""It can be used to discover the probable genotype frequencies in a population and to track their changes from one generation to the next

Godfrey Hardy (1877-1947)

Wilhelm Weinberg (1862-1937)

Evolution can be defined as a change in the frequencies

of alleles in the gene pool of a population over time

Images: Behavioral Sciences Department, Palomar College; http://anthro.palomar.edu/synthetic/synth_2.htm

To determine expected genotypic frequencies

Example: •  If p = 0.45 , then q = 0.55 since p + q = 1 p2 + 2pq + q2 = 1 (0.45)2 + 2 (0.45)(0.55) + (0.55)2 = 1

0.2025 + 0.495 + 0.3025 = 1

Using Hardy-Weinberg

These represent the expected genotypic frequencies if the population is in genetic equilibrium

• p2 = 20.25% • 2pq = 49.5% • q2 = 30.25%

CSHL DNA Learning Center Allele Server

Biostatistics Activity #2 - all online

page 20 in Teacher Guide

Chi Square Analysis Are our expected genotype frequencies similar to

the actual class frequencies?

This analysis will compare our group against a predicted population for our group, to see how well our group fits the theoretical prediction

How to interpret the results: Low values of chi square indicate that this population is close to the prediction High values indicate that this population is far from the prediction and suggest that this population's alleles do not fit the prediction model

Alu Global Distribution and Population Genetics

Reference Genotype Frequencies # Group + -33 Sardinian (Marrubiu) 0 134 Sardinian (Ollolai) 0 125 Nigerian 0.09 0.9110 German 0.1 0.913 Hungarian 0.12 0.8827 Papua New Guinea 0.14 0.863 Australia Aborigine 0.15 0.8532 Sardinian (Aritzo) 0.17 0.837 Euro-American 0.18 0.8211 Greek, Cyprus 0.18 0.8239 Syrian 0.18 0.8228 Papua New Guinea, Costal 0.19 0.811 African American 0.2 0.819 !Kung (''Bushmen'') 0.2 0.838 Swiss 0.2 0.85 Cajun 0.21 0.799 French 0.23 0.7717 Italian 0.24 0.7624 Nguni (Southern Africa) 0.24 0.7630 Pygmy (Central African Republic) 0.26 0.744 Breton (France) 0.27 0.7335 Sardinian (San Teodoro) 0.27 0.732 Alaska Native 0.29 0.7136 Sotho (Southern Africa) 0.29 0.7116 Indian Muslim 0.3 0.726 Pakistani 0.3 0.742 United Arab Emirates 0.3 0.729 Pushtoon (Afgani) 0.33 0.6731 Pygmy (Zaire) 0.35 0.6512 Hispanic American 0.51 0.4914 India Christian 0.52 0.4815 India Hindu 0.52 0.4823 Mvskoke (Seminole) 0.53 0.4737 South India 0.56 0.4441 Turkish, Cyprus 0.58 0.4222 Moluccas (Indonesia) 0.69 0.3121 Maya (Central America) 0.7 0.320 Malay 0.72 0.288 Filipino 0.8 0.218 Java 0.84 0.166 Chinese 0.86 0.1440 Taiwanese 0.9 0.143 Yanomamo (Amazon) 0.94 0.04

Since not everyone has the PV92 insert, it must have arisen after the initial human population began growing Where did the Alu insert originate?