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Introduction to Genetics and Genomics

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Terry Braun

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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

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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)

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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.

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Example -- genotypes1 1 1 1

1 1 1 1

Pedigreemalefemaleparentsoffspring

1 2 3 4

1 4 2 4

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).

uninformative heterogeneous

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What a marker looks like in the Genome

Geneticists assign numerical values to different versions of markers

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Sources of Markers in the Genome

• duplications• unequal homologous recombination• slippage and errors during DNA

duplication

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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)

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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)

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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)

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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

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Exponential Nature of Reaction

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Sequencing Reaction

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Automated

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Components of the Reaction

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DNA polymerase (Taq) and Synthesis

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Animations

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

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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)

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Marker GATA50G06/D15S643, Genotypes, and primers –

133101: 215, 197133102: 219, 215

Genomic chr15 :

ttctgctctt ttgtctaaaa tgtcagtcta aatccttact tgtaattgtg 57501064ccctactttg ccgttgctgc ctggctatac cttgtattta ttgctggcct 57501114ATACCTGGAG TCCTTGGTCC ttcttgggaa aaagtattga ggttttaaag 57501164ctcttatcct tggggacaga ttaaaccctt aaactatcta tctgtctgtc 57501214tgtctgtcta tctatctatc tgtctatcta tctatctatc tatctatcta 57501264tctatctatc tatctatcta cctacctaac tacctaccaa aaaaGCATTG 57501314AGGTTTTAAA GCTGTTatcc ttggggacag attaaaccct caaccctcta 57501364tctatctatc tatctatcta tctatctatc tatctatcta tctatctatc 57501414atctgtcacc 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=800http://research.marshfieldclinic.org/genetics/genotypingData_Statistics/genotypes_referenceIndividuals.asp

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Marker GATA50G06/D15S643, Genotypes, and primers –

133101: 215, 197133102: 219, 215

Genomic chr15 :

ttctgctctt ttgtctaaaa tgtcagtcta aatccttact tgtaattgtg 57501064ccctactttg ccgttgctgc ctggctatac cttgtattta ttgctggcct 57501114ATACCTGGAG TCCTTGGTCC ttcttgggaa aaagtattga ggttttaaag 57501164ctcttatcct tggggacaga ttaaaccctt aaactatcta tctgtctgtc 57501214tgtctgtcta tctatctatc tgtctatcta tctatctatc tatctatcta 57501264tctatctatc tatctatcta cctacctaac tacctaccaa aaaaGCATTG 57501314AGGTTTTAAA GCTGTTatcc ttggggacag attaaaccct caaccctcta 57501364tctatctatc tatctatcta tctatctatc tatctatcta tctatctatc 57501414atctgtcacc tattta

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Genome to Gene Sequence

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

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Basis for Inheritance of Disease: Examples

Aa Aa

A a

A

a

AA Aa

Aa aa

1/2 1/2

1/2

1/2

1/4 1/4

1/4 1/4

P(AA) = 1/4P(Aa) = 1/2P(aa) = 1/4

Aa Aa AA AA Aa

A from mom/dad?a from mom/dad?

Pedigreemalefemaleparentsoffspring

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Examples234 238 232 238

234, 238 238, 238 238, 232 234, 232234, 238

1 2322 2343 2364 2385 2406 242

2 4 1 4

Note that the lawyers for OJ Simpson argued that "recoded" allele numbers increased the likelihood of contamination and false identification.

If you "genotype" an individual at enough markers, you can calculate the probability of uniquely identifying an individual.

1 2342 2363 2384 2405 242

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Examples

Affected individuals

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ExamplesDominant model

Geneticists then look for genes that mimic this pattern of inheritance

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ExampleRecessive model.Very unlikely, because "founders"marrying in also carry the disease,which by definition is a raregenetic disorder.

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BBS4 Pedigree

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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???

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...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

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Bardet-Biedl Syndrome (BBS)• Obesity

– Diabetes/ hypertension

• Retinopathy• Hypogenitalism• Polydactyly• Mental Retardation• Renal Anomalies• Heart defectsRare disorder, but common phenotypes

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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

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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

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BBS4 Deletion (by PCR)Example of Usage

exons 3 4

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Molecular Genetics

• Not covered– molecular details of DNA duplication

• continuous replication, discontinuous, Okazaki fragments, etc.

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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?)

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Leber Optic Atrophy

• LHON– mid-life, central vision loss– caused by missense mutations in mtDNA– generally familial

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• Evolution of the mitochondrial genome and origin of eukaryotic cells

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END

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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.

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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

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Expected allele frequenciesDeviations from distributionmay indicate special cases.

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H-W

• f(AA) = p2

• f(Aa) = 2pq• f(aa) = q2

• (p+q)2

• (p2 + q2 + r2 + 2pq + 2pr + 2qr)= (p+q+r)2

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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 196marker2: AA 2 AB 37 BB 360marker3: AA 64 AB 144 BB 192

Which maker is suspicious?

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Will return to Linkage in Later Lectures

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