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INBRE High School Genomics 2013 2INFINITE VARIATIONS.

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Presentation on theme: "INBRE High School Genomics 2013 2INFINITE VARIATIONS."— Presentation transcript:

1 INBRE High School Genomics 2013 2INFINITE VARIATIONS

2 Genomics is driving a revolution in Genetics: DNA sequencing of 14,002 individuals for 202 drug metabolism genes rare variants occur every 17 bp (rare = 1 or 2 SNV in 14,000) over 50 variants per 1000 bases (NS 1 kb) are known (tip of iceberg) In summary: Each of us likely within our genomes 35 Nonsense mutations >100 variants that cause loss of protein function TRUE individual variation for evolution/selection 3INFINITE VARIATIONS

3 4 PersonalPersonal

4 Pre-Genomic Discovery The pre-Genomic Era September 1, 1989: Three papers are announced but embargoed in Science Magazine describing the causative gene in Cystic Fibrosis September 8, 1989 INFINITE VARIATIONS5

5 Does the Human Genome Exist? The original reported genome is not from one person, it is a composite of several donors. Donors were healthy at time but carry variants (10’s of thousands) some may carry disease variants We are all individual type specimens with INFINITE VARIATIONS. INFINITE VARIATIONS 6 It turns out to be a total F#@*&!ng mess. So instead of having a linear icon representing human biology, the most potent symbol now is the hairball.” Misha Angrist, author Here is a Human Being

6 Punnett Square is visual representation of Mendelian Inheritance 7INFINITE VARIATIONS

7 Course Outline Bioinformatics Exercises NCBI Gene Information Genome Browsers DNA sequence analysis Human SNPs Molecular Laboratory Exercises DNA Extractions Standard PCR Restriction Digests DNA sequencing Allele Specific PCR SNP genotyping 8INFINITE VARIATIONS

8 Bioinformatics Exercises Introduction of genome browsers 9INFINITE VARIATIONS


10 SNP Discovery and Description 11INFINITE VARIATIONS

11 Laboratory Work Modern Techniques Allele Specific PCR (high through-put) Uses fluorescence Traditional Techniques DNA Extractions Restriction Digests Newer Techniques PCR Second Generation Sequencing 12INFINITE VARIATIONS Experiment Block 1: Using PCR to Genotype CYP2C19, OXTR and ACE. Variants: indel, SNP ID by restriction digest Experiment Block 2: Using PCR and DNA Sequencing to Genotype, ACTN3, FUT2, AR, APOA5. Variants:, SNPs Experiment Block 3: Using Fluorescent Beacons to Genoptype allelic PCR of ACHOO, ACTN3

12 Variation types Single Nucleotide Polymorphism (SNP) is a point mutation Indel: insertion or deletion mutation in DNA sequence, in coding DNA sequence, unless the insertion or deletion occurs in a factor of 3 a frameshift mutation occurs. Frameshift Mutation: an insertion or deletion mutation that alters the reading frame of coding DNA sequence, frameshifts occur when the indel is not a factor of 3 Point Mutation: a mutation where one nucleotide is substituted for another, there is no addition of nucleotides relative to length of sequence. Point mutations can be categorized functionally: Nonsense mutations code for a stop, which can truncate the protein Missense mutations code for a different amino acid Copy Number Variants CNVs reflect quantitative changes in the genome (more or less copies of a gene or DNA region) INFINITE VARIATIONS13

13 CYP2C19 Cytochrome p450 gene Metabolizes hormones (e.g. estrogens) Metabolizes numerous drugs  Prilosec (anti-ulcer)  Plavix (clopinogel) (anti-platelet) FDA genotyping  Diazepam (anti-epileptic) Highly expressed in the Liver At least 17 known human alleles with differing drug metabolism phenotypes Three general human groups are widely recognized:  Poor metabolizers,  Medium metabolizers,  Extensive/Rapid metabolizers 14INFINITE VARIATIONS

14 CYP2C19 Phenotype & Genotype Frequency in Different Populations Three general human phenotypes are widely recognized: PopulationWhite %Black %Asian % Rapid metabolizers 1*/1* 746638 Medium metabolizers 1*/2* or 1*/3* 262950 Poor metabolizers 2*/2* 2*/3* 3*/3* 2414 15INFINITE VARIATIONS

15 Restriction endonucleases 16INFINITE VARIATIONS

16 PCR Amplification of CYP2C19 Exon 4 and Exon 5 SNP ID The “old fashioned” way Standard PCR Standard PCR Digested Using Restriction Digest of PCR products 17INFINITE VARIATIONS

17 ABI 3130XL 16-capillary automated DNA sequencer (with Christine Smith) Second generation technology 18INFINITE VARIATIONS

18 CYP2C19 Exon 4 SNP ENST00000464755ENST00000464755:1399G>A 19INFINITE VARIATIONS

19 CYP2C19 Exon 4 SNP Raw Sequence Heterozygote Individual Wildtype Individual 20INFINITE VARIATIONS

20 Actin binding protein Two versions in human ACTN2 and ACTN3  ACTN2 expressed in all muscle fibers  ACTN3 restricted to type 2 (fast twitch) fibers  Redundancy in function likely  ACTN3 likely has accessory function One SNP appears to be correlated with athletic performance R577X an arginine to stop codon Frequency of 577XX varies from ~1% to 25% ACTN3  Actinin 3 gene 21INFINITE VARIATIONS


22 Polymerase Chain Reaction Revolutionized Biology in the late 1980’s Dependent on themo-stable DNA polymerase Taq, from extreme thermophile bacteria A three cycle reaction series –Denaturation: breaks apart ds DNA ‘template’ –Annealing: anneals primers to DNA template –Extension: DNA polymerase builds DNA copies 23INFINITE VARIATIONS

23 Polymerase chain reaction (PCR) 24INFINITE VARIATIONS

24 The “modern” way KASPAR animation 25INFINITE VARIATIONS

25 ACHOO Results Photic Sneeze response NO DNA Control 5551 52 56 50 63 54 62 57 53 equi Genotype TT Genotype CT Genotype CC


27 Investigating your own Genome Biology 28INFINITE VARIATIONS

28 What does all this mean 1.Know that SNP genotyping and some genome sequencing results provide probabilistic data. (3.2% versus 2.4%; 33% risk) 2.It is the exception, rather than rule, that a genetic marker is directly linked to a health outcome ( e.g. Huntington’s Disease) 3.“Above average” and “below average” risk from a SNP test must be interpreted in the context of other risk factors. (Low risk for heart disease does not mean one won’t get heart disease because it is a very common disease in the U.S. Above average risk for MS will still mean low risk as MS is much less common than heart disease.) 4.Family and other health histories are still of paramount importance 5.Behavior is still paramount (smoking cessation, physical activity, diet etc.) 6.Science is rapidly changing, advancing; INDIVIDUAL Genomes are here to stay INFINITE VARIATIONS29 INFINITE VARIATIONS



31 Variant Assay Types INFINITE VARIATIONS32 Experiment Block 1Experiment Block 2Experiment Block 3

32 Variations typed in Lab INFINITE VARIATIONS33

33 Biology Review 1 34INFINITE VARIATIONS

34 Biology Review 2 35INFINITE VARIATIONS

35 Sequencing amplified and purified DNA 36INFINITE VARIATIONS


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