1. Introduction to Genetics and Genomics 51:123 Terry Braun

2. Outline • Basic Mendelian Genetics • Mendel’s laws • independent assortment • independent segregation • mitosis and meiosis • PCR and markers • dominant/recessive and pedigrees • genotype and phenotype • alleles • Basic molecular genetics • DNA • RNA • proteins • Central Dogma • genes and gene structure • cells and chromosomes Principles of Genetics, Tamarin, Human Molecular Genetics 2, Strachan and Read

3. KeyTerms • marker – a region of the genome that may often be uniquely identified and distinguished between individuals • minisatellite – a type of marker that varies in length from 14 to 100 nucleotides • microsatelite – a type of marker that is very short (2, 3, 4, 5, 6 nucleotides) -- aka STRP's (short tandem repeat polymorphisms) • polymorphism – a sequence variation • SNP -- single nucleotide polymorphism • polymerase chain reaction (PCR) – a reaction that mimics DNA duplication in meiosis (aka DNA amplification) (Kary Mullis) • DNA polymerase – a molecule that is essential for DNA duplication (and PCR) • primer – a piece of DNA that is essential for starting DNA replication (and PCR) • genotype – the genetic state of an individual (typically represented by a marker)

4. Genetic Marker • A genetic marker allows for the observation of the genetic state at a particular genomic location (locus). • A genotype is the measured state of a genetic marker. • A tool for observing inheritance patterns (Mendel's rules and meiosis) • May never be feasible to sequence cases directly, however the current cost is decreasing • An “informative” marker is often “heterogeneous, or “polymorphic” and enables the observation of the inheritance of genetic material.

5. Example -- genotypes Pedigree male female parents offspring 1 2 3 4 1 1 1 1 2 4 1 4 1 1 1 1 uninformative heterogeneous These labels (markers) are a measure of the genetic state of each individual. Recall from "Rule of Segregation", offspring get one gene from each parent. Markers are not genes, but they are regions on chromosomes (meiosis).

6. What a marker looks like in the Genome Geneticists assign numerical values to different versions of markers

7. Sources of Markers in the Genome • duplications • unequal homologous recombination • slippage and errors during DNA duplication

8. Duplicating DNA – to Use Markers to "Probe" Genomes of Individuals • mitosis is process that copies DNA in biology • the first step is to "unzip" the 2 strands of the double helix (DNA) • an enzyme called DNA polymerase makes a copy by using each strand as a template • two other components • nucleotides (A, G, T, C) (A-T, G-C, etc) • a short stretch of DNA called a "primer" (to prime the process)

9. PCR – Polymerase Chain Reaction • PCR is a process that copies DNA exponentially • mimics the process by organisms, but in vitro (in a test tube) • relies on the ability of DNA-copying enzymes to remain stable at high temperatures • Necessary components (in a vial) • piece of DNA to be copied • large quantities of four nucleotides • large quantities of primer sequence • DNA polymerase (Taq – named for Thermus aquaticus, a bacterium that lives in hot springs)

10. PCR Reaction • The reaction can be carried out entirely in a vial simply by changing the temperature • separate the 2 strands (in DNA) • heat to 75-90 C (165 F) for 30 seconds • this "melts" the DNA apart – the base pairing comes undone • "anneal" the primers • primers cannot bind to the template strands at such high temp – cooled to 55 C for 20 seconds • make complete copy of template (and thus new templates for the next cycle) • Taq polymerase works best at 75 C (hot springs) • nucleotides are added (complement – if template has A, T is added, etc)

11. PCR Reaction • Three steps • separation of strands • annealing of primers to template • synthesis of new strands • Takes approx. 2 minutes • Each reaction is carried out in the same vial, and after every cycle, each piece of DNA is duplicated (exponential copying) • Cycle can be repeated 30 times (2^30 = 1,073,741,824) • 1 million copies can be made in approximately 3 hours from a single copy of DNA • this is why very minute samples can be used to identify individuals in crime scene investigations • Valuable tool to multiply unique regions of DNA so they can be detected in LARGE genomes • Note, we need to know the flanking sequence to be able to design primers • Also, this flanking sequence needs to be unique otherwise the reaction could amplify sequence from multiple regions of the genome

12. Exponential Nature of Reaction

13. Sequencing Reaction

14. Automated

15. Components of the Reaction

16. DNA polymerase (Taq) and Synthesis

18. Animations • http://allserv.rug.ac.be/~avierstr/principles/pcrani.html

19. Markers – the early days • Prior to the HGP, markers were (and still are) valuable tools for observing inheritance patterns • Investigators consumed considerable time and resources identifying markers • Some markers were observed in a test group of individuals to asses quality, and heterogeneity. • CEPH (Centr d'Etude du Polymorphisme Humain) • Affymetrix SNP Chip -- 500,000 SNPs (~$450 -- 2007)

20. Marker GATA50G06/D15S643, Genotypes, and primers – 133101: 215, 197133102: 219, 215 Genomic chr15 : ttctgctctt ttgtctaaaa tgtcagtcta aatccttact tgtaattgtg 57501064 ccctactttg ccgttgctgc ctggctatac cttgtattta ttgctggcct 57501114 ATACCTGGAG TCCTTGGTCC ttcttgggaa aaagtattga ggttttaaag 57501164 ctcttatcct tggggacaga ttaaaccctt aaactatcta tctgtctgtc 57501214 tgtctgtcta tctatctatc tgtctatcta tctatctatc tatctatcta 57501264 tctatctatc tatctatcta cctacctaac tacctaccaa aaaaGCATTG 57501314 AGGTTTTAAA GCTGTTatcc ttggggacag attaaaccct caaccctcta 57501364 tctatctatc tatctatcta tctatctatc tatctatcta tctatctatc 57501414 atctgtcacc tattta http://genome.ucsc.edu/cgi-bin/hgc?hgsid=76756345&o=57501058&t=57501337&g=stsMap&i=GATA50G06&c=chr15&l=57401058&r=57601337&db=hg18&pix=800 http://research.marshfieldclinic.org/genetics/genotypingData_Statistics/genotypes_referenceIndividuals.asp

21. Marker GATA50G06/D15S643, Genotypes, and primers – 133101: 215, 197133102: 219, 215 Genomic chr15 : ttctgctctt ttgtctaaaa tgtcagtcta aatccttact tgtaattgtg 57501064 ccctactttg ccgttgctgc ctggctatac cttgtattta ttgctggcct 57501114 ATACCTGGAG TCCTTGGTCC ttcttgggaa aaagtattga ggttttaaag 57501164 ctcttatcct tggggacaga ttaaaccctt aaactatcta tctgtctgtc 57501214 tgtctgtcta tctatctatc tgtctatcta tctatctatc tatctatcta 57501264 tctatctatc tatctatcta cctacctaac tacctaccaa aaaaGCATTG 57501314 AGGTTTTAAA GCTGTTatcc ttggggacag attaaaccct caaccctcta 57501364 tctatctatc tatctatcta tctatctatc tatctatcta tctatctatc 57501414 atctgtcacc tattta

22. Genome to Gene Sequence Markers are typically NOT genes, however they may reside in the genome relatively close to a gene.

23. Basis for Inheritance of Disease: Examples Aa Pedigree male female parents offspring Aa Aa Aa AA AA Aa 1/2 1/2 A a A from mom/dad? a from mom/dad? P(AA) = 1/4 P(Aa) = 1/2 P(aa) = 1/4 1/2 1/4 1/4 A AA Aa 1/2 1/4 1/4 Aa aa a

24. Examples • 234 • 236 • 238 • 240 • 242 • 232 • 234 • 236 • 238 • 240 • 242 234 238 232 238 1 4 2 4 3 234, 238 238, 232 234, 232 234, 238 238, 238 If you "genotype" an individual at enough markers, you can calculate the probability of uniquely identifying an individual. Note that the lawyers for OJ Simpson argued that "recoded" allele numbers increased the likelihood of contamination and false identification.

25. Examples Affected individuals

26. Examples Dominant model Geneticists then look for genes that mimic this pattern of inheritance

27. Example Recessive model. Very unlikely, because "founders" marrying in also carry the disease, which by definition is a rare genetic disorder.

28. BBS4 Pedigree

29. Monogenic and Polygenic Diseases • monogenic (Mendelian) -- one gene • “simple” (dominant and recessive) Mendelian inheritance • direct correspondence between one gene mutation and one disorder • majority of disease genes found are monogenic • polygenic -- (complex) multiple genes • heterogeneity – disease caused by multiple genes • epistasis – disease caused by multiple interacting genes • obviously finding these is harder -- but why???

30. ...Mongenic and Polygenic Diseases • phenocopy • reduced penetrance • Example -- sickle cell anemia • “classic” recessive disorder • defect in red blood cells (hemoglobin) • but… infant hemoglobin gene can “leak” • wide range of phenotypes

31. Bardet-Biedl Syndrome (BBS) • Obesity • Diabetes/ hypertension • Retinopathy • Hypogenitalism • Polydactyly • Mental Retardation • Renal Anomalies • Heart defects Rare disorder, but common phenotypes

32. Molecular Analysis of BBS • BBS1 - 11q13 Novel* • BBS2 - 16q22 Novel* • BBS3 - 3p13 • BBS4 - 15q21 Novel†, TPR Repeats • BBS5 - 2q31 • BBS6 - 20p12 Type II Chaperonins • BBS7 - 4q27 Novel* • BBS8 - 14q31 Novel†, TPR Repeats *,† - Some Similarity

34. Some Useful Properties of DNA • fragments of DNA have a minute negative charge • if you apply an electric field to DNA in a matrix, it will migrate to the positive pole • DNA is a linear molecule, but it tends to fold up (similar to a knot) • this bound up molecule of DNA will have a unique cross-sectional area profile that is dependent on its sequence • Gel electrophoresis – DNA is placed in a polyacrylamide gel and a voltage is applied • polyacrylamide gel and pool analogy • applied charge will cause DNA to migrate dependent on its size, and its sequence

35. BBS4 Deletion (by PCR)Example of Usage exons3 4

36. Molecular Genetics • Not covered • molecular details of DNA duplication • continuous replication, discontinuous, Okazaki fragments, etc.

37. Genome – so now we know where it comes from biologically – at least most of it • mitochondria • organelle of eukaryotes • number varies per cell – 10 to 10K • human mitochondria is 16,569 nts • mostly coding (no introns???) • duplex strand and circular • inherited maternally only • consequences • mito thought to be originally free-living bacteria • origins (one or multiple events?)

38. Leber Optic Atrophy • LHON • mid-life, central vision loss • caused by missense mutations in mtDNA • generally familial

39. Evolution of the mitochondrial genome and origin of eukaryotic cells

40. END

41. Another Marker? BRCA1-A good predictive marker of drug sensitivity in breast cancer treatment? * Mullan PB, * Gorski JJ, * Harkin DP. Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland, BT9 7AB, United Kingdom. There are currently only two predictive markers of response to chemotherapy for breast cancer in routine clinical use, namely the Estrogen receptor-alpha and the HER2 receptor. The breast and ovarian cancer susceptibility gene BRCA1 is an important genetic factor in hereditary breast and ovarian cancer and there is increasing evidence of an important role for BRCA1 in the sporadic forms of both cancer types. Our group and numerous others have shown in both preclinical and clinical studies that BRCA1 is an important determinant of chemotherapy responses in breast cancer. In this review we will outline the current understanding of the role of BRCA1 as a determinant of response to DNA damaging and microtubule damaging chemotherapy. We will then discuss how the known functions of this multifaceted protein may provide mechanistic explanations for its role in chemotherapy responses.

42. Hardy-Weinberg Equilibrium • Rule that relates allelic and genotypic frequencies in a population of diploid, sexually reproducing individuals if that population has random mating, large size, no mutation or migration, and no selection • Assumptions • allelic frequencies will not change in a population from one generation to the next • genotypic frequencies are determined in a predictable way by allelic frequencies • the equilibrium is neutral -- if perturbed, it will reestablish within one generation of random mating at the new allelic frequency • Ideal case

43. Expected allele frequencies Deviations from distribution may indicate special cases.

44. H-W • f(AA) = p2 • f(Aa) = 2pq • f(aa) = q2 • (p+q)2 • (p2 + q2 + r2 + 2pq + 2pr + 2qr)= (p+q+r)2

45. Use of H-W • All other things being equal, we can "expect" that the distribution of genes in a subset of a population would be represented by the distribution of genes in the population • Deviations from this expected distribution is evidence of selection or enrichment • Association – when a specific variation of a gene (allele) is correlated with a phenotype (or disease, or trait) more frequently than you would expect by H-W • also called Linkage Disequilibrium (since genes are normally in equilibrium) Often used to evaluate validity of an assay. For example, let us say that I genotype 400 people at a marker with 2 alleles (A and B). I observe the following genotypes: marker1: AA: 36 AB 168 BB 196 marker2: AA 2 AB 37 BB 360 marker3: AA 64 AB 144 BB 192 Which maker is suspicious?

46. Will return to Linkage in Later Lectures