Introduction to Genetics and Genomics

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Introduction to Genetics and Genomics

• 51123
• Terry Braun

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Outline

• Basic Mendelian Genetics
• Mendels 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 -- 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).
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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 (230
  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/pcra
  ni.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 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?hgsid76756345o57501058t57501337
gstsMapiGATA50G06cchr15l57401058r57601337
dbhg18pix800 http//research.marshfieldclinic.
org/genetics/genotypingData_Statistics/genotypes_r
eferenceIndividuals.asp
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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
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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
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
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Examples

• 234
• 236
• 238
• 240
• 242

1. 232
2. 234
3. 236
4. 238
5. 240
6. 242

234 238
232 238
1 4
2 4
3
238, 232
234, 232
234, 238
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.
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Examples
Affected individuals
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Examples
Dominant model
Geneticists then look for genes that mimic this
pattern of inheritance
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Example
Recessive model. Very unlikely, because
"founders" marrying in also carry the
disease, which by definition is a rare genetic
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 defects

Rare 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 frequencies Deviations from
distribution may indicate special cases.
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H-W

• f(AA) p2
• f(Aa) 2pq
• f(aa) q2
• (pq)2
• (p2 q2 r2 2pq 2pr 2qr) (pqr)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 196
• marker2 AA 2 AB 37 BB 360
• marker3 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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