Introduction to PCR. Kristen Wolslegel Manager, Education Programs BABEC

Transcription

1 Introduction to PCR Kristen Wolslegel Manager, Education Programs BABEC

2 What is DNA? It encodes within its structure the hereditary information that determines the form and function of all known living organisms. 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

3 What is DNA made of? DNA consists of building blocks called nucleotides 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 Image: SCFBIO

4 2 Types of Nitrogenous Bases (4 in total) Pyrimidines: 6-member ring Cytosine & Thymine Purines: fused 5 & 6 member rings Adenine & Guanine

5 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

6 DNA has two strands with bases paired in the middle

7 DNA Replication Animation Molecular Cell Biology, Lodish et. al. 4th ed.

8 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 Why? 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

9 DNA Learning Center Videos

10 Primers Primer 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

11 DNA Polymerase from Thermus aquaticus! A thermostable DNA polymerase Can function at high temperatures Isolated from a thermophilic bacteria that could withstand 95 o C Discovered in 1965 in the hot springs of Yellowstone National Park Commonly known as Taq Images: Canadian Agricultural Board /06/25/hydrothermal_vent_colonization/

12 Deoxynucleotide-triphosphates: dntps The PCR Building Blocks A dntp mix contains equal amounts of : datp dttp dgtp dctp

13 The PCR Cycle Denature: o C Anneal: o C Extension: 72 o C 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

14 After 30 cycles, DNA is amplified over a billion-fold! Target sequence Cycle 1 Cycle 2 Cycle 3 Cycle Relative Amount , ,048, ,073,741,824

15 PCR vs. Cellular DNA Replication What are we copying? How do we separate the DNA? Polymerase Chain Reaction DNA Heat Cellular DNA Cell Replication DNA Enzymes What is doing the copying? How do we fish out the sequence? Taq polymerase Primers Human polymerase Primers What does the work? Thermal cycler Cell

16 PCR in Medicine Diagnosis of diseases: Cancer, microbial & viral infections Blood type testing Detection of heritable/genetic diseases (amniocentesis)

17 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

18 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

19 PCR in Anthropology & Evolutionary Biology Phylogenic analysis of DNA from ancient sources (Neanderthals, Mammoths) Analysis of ancient humans Study evolutionary relationships Image: Georgia Perimeter College / /chap_tut/chaps/chapter17-05.html Image: Penn State

20 Agarose Gel Electrophoresis

21 Agarose Gel Electrophoresis well #1 well #2 well #3 _ DNA Ladder large DNA fragments small DNA fragments Dye is added to give the DNA color + Electron micrograph of an agarose gel

22 Alu is DNA that can move! " " 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.

23 How is Alu passed on from our parents? Alu+/Alu- Alu-/Alu- Alu+/Alu- Alu+/Alu- +/- +/- -/- -/- +/+ +/- -/+ -/- Alu may be present or absent on each of your paired chromosomes, creating two possibilities (+ and ) Inherited from both parents +/+ Inherited from one parent.. +/- These are called alleles Not inherited -/-

24 The Alu PV92 DNA sequence amplified by PCR chromosome 16 without insert Forward primer chromosome 16 with insert Forward primer PCR Target 415 bp (-) PCR Target Alu (300 bp)?? (+) Reverse primer Reverse primer Student #1 Student #2 Student #3 715 base pairs 415 base pairs +/+ +/- -/-

25 Alu tells us about human migration 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

26 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"

27 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

28 Calculating Allele Frequencies Allele Frequency: the percent of a particular allele within a population Frequency of (+) = # of (+) alleles # of students Frequency of (-) = # of (-) alleles # of students There are twice as many total alleles as there are people

29 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

30 Calculating Genotype Frequencies The Hardy-Weinberg Equation p 2 + 2pq + q 2 = 1 p q p q pp pq pq qq +/+ = p 2 +/- = 2pq -/- = q 2

31 The Hardy-Weinberg Principle Evolution can be defined as a change in the frequencies of alleles in the gene pool of a population over time Godfrey Hardy ( ) Wilhelm Weinberg ( ) 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 Images: Behavioral Sciences Department, Palomar College;

32 Using Hardy-Weinberg To determine expected genotypic frequencies Example: If p = 0.45, then q = 0.55 since p + q = 1 p 2 + 2pq + q 2 = 1 (0.45) (0.45)(0.55) + (0.55) 2 = = 1 p 2 = 20.25% 2pq = 49.5% q 2 = 30.25% These represent the expected genotypic frequencies if the population is in genetic equilibrium

33 CSHL DNA Learning Center Allele Server Biostatistics Activity #2 - all online page 20 in Teacher Guide

34 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

35 Alu Global Distribution and Population Genetics

36 Reference Genotype Frequencies # Group Sardinian (Marrubiu) Sardinian (Ollolai) Nigerian German Hungarian Papua New Guinea Australia Aborigine Sardinian (Aritzo) Euro-American Greek, Cyprus Syrian Papua New Guinea, Costal African American !Kung (''Bushmen'') Swiss Cajun French Italian Nguni (Southern Africa) Pygmy (Central African Republic) Breton (France) Sardinian (San Teodoro) Alaska Native Sotho (Southern Africa) Indian Muslim Pakistani United Arab Emirates Pushtoon (Afgani) Pygmy (Zaire) Hispanic American India Christian India Hindu Mvskoke (Seminole) South India Turkish, Cyprus Moluccas (Indonesia) Maya (Central America) Malay Filipino Java Chinese Taiwanese Yanomamo (Amazon) Since not everyone has the PV92 insert, it must have arisen after the initial human population began growing Where did the Alu insert originate?