Genotyping

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Genotyping

• Broadly defined
• Identifying human genetic variation.
• More restricted sense
• Laboratory tests for certain kinds of human
  genetic variation Polymorphisms. A key part of
  Genomics research today.

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Quiz What is Genomics?

• The study of very small, elf-like people.
• Something I dont need to know about
• so why bother?
• C. The scientific study of the sequence and
• functioning of the human genome and
• other living organisms.

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Basic Definitions

• Genetics is the study of single genes and their
  effects on health

• Genome refers to all of the genetic material
  (DNA) belonging to an organism

• Genomics is the study of all the genes in the
  genome, including their interactions with
  environmental factors

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Definition Polymorphism
Variations in DNA sequence (substitutions,
deletions, etc) that are present at a frequency
greater than 1 in a population. They have a WEAK
EFFECT and sometimes no effect at all. They are
ancient and COMMON.
Definition Mutation

• Variations in DNA sequence (substitutions,
  deletions, etc) that are present at low frequency
  in a population (e.g. 1/10,000).
• They often reduce function and have a STRONG
  EFFECT.
• They may have recent origin and are RARE.

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Example Polymorphism
MCIR Melanocortin Receptor Red hair, freckles,
pale skin
Example Mutation

• BRCA1 and BRCA2
• Breast and ovarian cancer in high risk families

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Screening vs. Genotyping

• Screening for mutations
• Searching for one of many possible mutations in
  a large region of DNA.
• Example DNA sequencing
• Genotyping for polymorphisms
• Looking for a specific variant at one location
• Single Nucleotide Polymorphisms SNPs

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SNPs

• Human beings differ on average every thousand
  base pairs of DNA by at least one base in the DNA
  sequence
• G to T
• A to C
• A to T
• etc.

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Chromosome Structures
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Inside the DNA structure

• adenine (A)
• thymine (T)
• cytosine (C)
• guanine (G)
• TACGCGTTAATCA
• ATGCGCAATTTAG

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Genes contain the directions which guide cell
function
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Genes the Environment

• Most diseases involve the interaction of both
  genes and environmental factors.
• Gene exposure to
• chemicals
• tobacco use
• nutritional habits
• alcohol or drug use

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How do we detect SNPs?

• Genotyping methods have been developed such as
  Taqman.
• Fast, cheap and reliable.

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TRADITIONAL GENOTYPING
A / A
B / B
A / B

• PCR RFLP
• 3 steps
• 1.Amplification
• 2. Digestion
• 3. Electrophoresis

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RFLP analysis on Agarose Gel
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High Throughput Genotyping Methods

• Taqman
• Light Cycler
• Syber Green
• HPLC
• Mass Spectroscopy
• Chips other solid phase platforms
• Beads

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Example of Genotyping Taqman

• One step genotyping method
• ABI Prism 7700 Sequence Detector
• 5 exonuclease assay (Taqman)

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TaqMan assay
Reporter dye
Quencher dye
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Taq Extends Upstream Primer
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Taq Digests Probe
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Bigger, better, faster!

• New formats ABI 7900 Sequence Detection System
• 96 well -gt 384 well plates
• Robotics
• Electronic data capture

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Genotyping Evolution

• Generation Then RFLP
• Generation Now Taqman
• Endpoint detection using ABI 7700 96-well
  plates.
• ABI 9700s for PCR amplification
• Assays-on-demand / Assays-by-design
• Real-time data capture.
• ABI 7900 384-well plates with robotics.
• Generation Next
• Multiplexing, e.g. Illumina platforms

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Generation Next

• Key feature is multiplexing
• Illumina
• Multiplex 1536 SNPs at a time using 96-well plate
  format.
• Bead platform. High resolution optical scanner.
• Late 2004 BeadChip with 100,000 SNPs.

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Illumina BeadStation 500G SNP Assay
Day 1
Day 3
A/A
A/G
G/G
Day 2
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LABORATORY OUTPUT

• One technician can conduct over 1,000 assays per
  day!
• TONS of data!
• Thousands of SNPs!
• Millions and millions of genotypes!

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Why such high throughput?

• Whole genome association studies using tag SNPs
  
• So-called haplotype tags (ht SNPs) used to
  identify constant chromosomal regions. Based
  on extensive linkage disequilibrium and the
  relatively recent common origins for all existing
  human populations.
• Some optimistic researchers think you can assay
  all
• 10 million SNPs in the human genome using
• 500,000 ht SNPs (Jan 2005 estimate).

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QUALITY CONTROL

• Laboratory Design
• Clean PCR Room, separate aliquotting space,
  offices for technicians
• Genotyping Protocols
• Positive and negative controls, 10 repeats
• Data Management
• Confidentiality/passwords, anonymous IDs.

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QUALITY CONTROL

• NCI SNP 500 project
• Assays designed ahead of time (ABI)
• Coriell positive controls (seaquenced)
• www.nci.snp500.gov
• Positive controls with every plate (batch)
• 10 repeats at random
• Repeat all no amps at least once.

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Methylation
Metabonomics
Cell-Cell Interactions
Genome
Genotype
Results of Laboratory Assay
mtDNA
Gene Expression
Everything Else
Proteome
Environment
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Know
Don't Know
Don't Even Know That We Don't Know