1 DNA sequencing Why? – Identifies OrganismsATGTTCTATCCCATTCATTTTGACGTTATTGTTGTTGGAGGAGGTCATGCTGGGACAGAAGCAGCTTTGGCTTCTGCTAGGATGCAATGTAATACGCTTTTGATTACTCATAATATTGATACTCTTGGACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGAAGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGATGAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCAAAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTTTCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCTTGTTATATTACCCATACTAATAACAAAACACATGAAATAGTTAGATCAAATTTATATCAGAGTCCTATGTATACAGGTTTAATAAAAGGAATAGCACCTCGGTATTGTCCATCTATAGAAGACAAAATAACTCGTTTTTCAGATCGTAATGCTCATCAAATTTTTTTAGAACCTGAAGGTTTGACAACACCTGAAGTATATCTTAATGGTATTTCTACTAGCTTACCTTTTTGTGTACAAATGCAAATGATTAAATCAATTCAGGGATTAGAGAATGCTTGTATAATTAGGCCAGGTTATGCTATTGAATACGATTTTTTTGATCCTCGTGATTTAAAATTAACATTAGAGAGTAAAATTATTTCTGGATTATTTTTTTCTGGTCAAATTAATGGTACTACTGGTTATGAAGAAGCAGCTGCCCAAGGATTATTGGCCGGAATTAATGCAGCTAGGTTTTCCAAAAATAAAGAAGGATGGTACCCTAGAAGAGATCAGGCATATTTAGGGGTGCTTGTAGATGATCTCTGTACACATGGAACAGAAGAACCTTATCGTATGTTTACTTCGCGTGCTGAATATCGTCTGTCTTTACGTGAGGATAATGCTGATTTAAGATTAACTGAAATCGCGCGACAGTTAGGTTTGATAGATGAGTCACGGTGGAAAGCTTTTTGTTGCAAAAAAGAAAACATTGAAAAAGAACGTCAGAGATTACGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAACACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAAGTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAATAAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTATAATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGACAAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTATCACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTAATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGGGTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAATAATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTATGTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAATAGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAAATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGTGTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGTGTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTCTATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACAACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTTGTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAATATTTTTTGAAAGGTGGTTTATGTCAGGAATCAAAGGCACTTCCCAAGAATATATTGGACATCATTTATATCATCTGCAATTTGATTTAAGTACTTTTTCGTTAGTGAGCTCGGAGAATACTTCTTCATTCTGGGTATTAAATGTAGATTCGATGTTTTTTTCAATATTATTGGCTACTTTATTTTTATTAATTTTCGGTCGTTTGGCTACAGTGGCAACTTATGCGGTTCCCACAAAACTGCAAGTGTTTATTGAGTTAGTAATATTGTTTATTGATAGCAATGTAAAAGATATGTTTCATGGTAAAAATAAACTAATTGCGCCATTATCTATGACGGTTTTTGTTTGGATTTTTTTAATGAATACCATGGATTTATTTCCCATAGATTTATTTCCTGCTATAGCTAAATTGTTAGGATTACCTGCTTTACGTGTTGTGCCGTCTGCCGATGTGAATATCACTTCTTCGCTAGCTTTAAATGTATTTGTACTTGTTATGTACTACAATATTTACGTTAATGGCGTTCATGGTTTTATTAAAGGACTGATGTATCATCCATTTAATCATCCAACATGTATTCCTATTAATTTTATTATTGAAGTTGTTAGTTTGTTATCTAAACCAGTATCACTTAGTCTTAGATTATTTGGCAATATGTATTCTGGTGAGTTGATTTTTATTTTGATATCTGGTTTATTACCATGGTGGGGACAATGGGTTTTAAATTTACCATGGGCTATTTTTCATATTTTAGTCGTTACATTACAGGCTTTTATTTTTATGGTTTTAACGGTGATTTATTTATCTACAGCCCATGACTCCTGTTAAAATGAATCATGTAATACAGAAGATTGCAGAGAGGTTATTATGGAACATTTAAATTTTGATATGTTATATATTGCTGCAGCAATAATGATGGGATTAGCAGCAATTGGAGCGGCTATCGGTATTGGCATCTTAGGTAGTAAATTTTTAGAGGGGGCTGCACGTCAACCAGATCTCATTCCGATCCTTCGAACTCAGTTTTTTATTGTTATGGGATTGGTTGATGCAATACCAATGATTACTGTAGGTCTTGGTTTGTATGTGATGTTTTCTGCGGTTTAACAATGAAAATTAATTGCATGTATTTATTATATTGATTATTAAATATCAGGATTTGCGCTGTGAATCTTAATGCAACAATATTAGGTCAAACTATTTCATTTGTTTTGTTTGTTTGGTTTTGTATGAAGTATGTGTGGTATCCATTTATATCTATTATTGAGAAACGCCAAAAAGAAATTTCTGATAATTTAGTTTCTGCTACTCATGCCAAAACAGAATCTGAGCGTGTCAATGCTGAAGCTTTGCTTTGTTTGAGACAGGCTCGGGTCAAAGCTCAAGAAATTATAAAACAAGCAAATAAATGTAAAATGCAAATAATTAATGAAGCTAAACATGAAGCTGAAAAGGAGCAAAGTAGAATTTTATCTCAAGCGCGAGAACAGATTATTTATGAAAGAAAACGTGTTACTGACGAATTAAGAAAGCAAATTAGCGAACTTGTAATTGAGGGTACAGAGAAAGTTATAGAACATTCTATAAATGAAATGATTGATATAGATCTTTTAAATAATATTATTAATACGTTGTCATATAAGGATTAGATGTCTAGTATGCTTGTGGTTGCGCGTACGTATGCTCAAGCTATATTTGATATAGCTGTAGAACAGAAAAATATAAACAAGTGGAAATCAGTTCTTGATTTATTTTCTGAGATTAGTCTAAATAGACTAGTACAATCTTTATTTTTTAGATGTTTAGAACCAAAAAGATTGTCAGATATATTTATTGCTATTTGTGAAGATTACCAAAAGAAACAAGTTGATACCTTCAGTAAAAATATAATCTATATTATGGCGGAAAATAATCGTTTATTATTATTACCAATTGTATTTAAAGAGTTTACTTATTTATGTTCTATATATGTTCATACTGTAGAAATAGAAATTATCTCTGCTTGGCCTTTGAAGTATAATCAGCTGAAAAAAATTACTGATATAATGGCTAAACGTTTATCTAAAACAGTGAATCCAGTACACAAAGTAGATAAAGATATATTGGCTGGTGTAATTATTCGTATTGGAGATACTGTGATTGATGGAAGTATACGTGGACGTATTTTTCGCTTAAATCACGTACTACAATCTTAATATTTTAACAGTAGTTAAAAGGTATAAACAAAAGATTATGCAATTAAATTCAAATGAAATTTCAGATAATGGAGAAGTTATTATTAATGATTTAAAGTTATTTTATAATAAAGCTAGACAAACTAAAATTACAGAAGAACTTACAGAAATTGTTTCAGGAGCTTCTGTAATATAAACTTAAAAGATAGTTAGAGGTATAATTAATATGAGTTCTGGAAAAATTGTCCAGGTTATTGGAGCGGTGGTTGATGTTGCGTTCAATCAAGATGTGGTACCGACTGTATACCATGCACTTGAGGTGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAACACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAAGTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAATAAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTATAATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGACAAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTATCACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTAATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGGGTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAATAATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTATGTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAATAGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAAATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGTGTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGTGTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTCTATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACAACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTTGTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAATATTTTTTGAAAGGTGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGAAGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGATGAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCAAAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTTTCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCT DNA sequencing Why? – Identifies Organisms Introduces Biological Machines How? – Dideoxy Sequencing Where? – Universities, MBL Societal Impact? – Forensics, Insurance DNA detective
2 Insect identificationReview of Project PCR DNA extraction Insect identification ? ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAG Bioinformatics DNA sequencing Gel electrophoresis ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCA
3 PCR (Polymerase Chain Reaction)1- Amplifies defined DNA sequences 2- Uses basic properties of the DNA replicating enzyme (DNA Polymerase) 3- DNA polymerization using defined DNA template, two defined primers, 4 dNTPs
4 DNA Polymerase Action
5 Basics of PCR Template DNA - the starting DNA of interest.DNA-based Technologies Lecture - Dr. Seth Bordenstein Basics of PCR Template DNA - the starting DNA of interest. High temperature denatures template DNA into single strands and synthetic sequences of ssDNA (20-30 nucleotides) serve as primers Two different primers are used to bracket the target gene to be amplified DNA polymerase copies the complimentary strand starting at the primer. In one cycle, two identical strands are made.
6 PCR - Ready Beads Small quantity of DNA PrimersDNA-based Technologies Lecture - Dr. Seth Bordenstein PCR - Ready Beads Small quantity of DNA Primers Buffered solution containing DNA polymerase Four base pairs of DNA Cofactor MgCl2 Small quantity of DNA Primers All in test tube Buffered solution containing DNA polymerase Four base Cofactor MgCl2
7 Two Key Innovations for Success of PCRDNA-based Technologies Lecture - Dr. Seth Bordenstein Two Key Innovations for Success of PCR Heat-stable DNA polymerase isolated from bacterium Thermus aquaticus which inhabits hot springs Polymerase remains active despite being heated many times DNA thermal cyclers – a computer that controls repetitive temperature changes required for PCR 70C hot springs in Yellowstone National Park Example of a thermal cycler from MJ Research
8 Temperature drives the reactionDNA-based Technologies Lecture - Dr. Seth Bordenstein Temperature drives the reaction
9 Target gene: 16S rDNA of WolbachiaDNA-based Technologies Lecture - Dr. Seth Bordenstein Target gene: 16S rDNA of Wolbachia
10 Importance of Ribosomes in Cell BiologyRibosomes are part of the machinery that synthesizes cell proteins Ribosomes are so important that they are ubiquitous and highly conserved
11 Molecular PhylogeneticsStep 1. Select a DNA region that is homologous, or similar across species due to common ancestry. Ribosomal RNA (rRNA) Ideal gene for phylogenetic studies because it : is an essential gene that is present in all organisms. is a common target for sequencing studies; large database for comparisons. contains sites that are relatively conserved (stems) and sites that are more free to vary (loops). Secondary structure of 16S rRNA in E. coli
12 Our goal: Determine which of your insects harbor Wolbachia?DNA-based Technologies Lecture - Dr. Seth Bordenstein Our goal: Determine which of your insects harbor Wolbachia? DNA extraction: PCR: Gel electrophoresis:
13 PCR and Pop Culture “Jurassic Park” and “CSI”DNA-based Technologies Lecture - Dr. Seth Bordenstein PCR and Pop Culture “Jurassic Park” and “CSI” Some fun PCR facts to share with your students: …PCR has been used to amplify DNA from… a preserved quagga (a zebra relative that became extinct 100 years ago) crime scenes (e.g., O.J.) Abducted children to find parents Prisoners leading to exonerations Mummies to determine gender, bacterial infections
14 How is this organism related to other species?K ? C A H F I J D B E G
15 DNA sequences provide characters that are:Numerous, discrete characters (A, T, C, G) Directly comparable across species Unlikely to change due to culture conditions Can be sampled without ever culturing (important for endosymbionts!) Molecular phylogenetics: Inference of evolutionary relationships based on molecular data ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTCAATACGCGTGCTCAGGCAGATAAAATATTA
16 2. Amplify and Sequence this region across isolates….ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGGTFTGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATTTAGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT Sequence the PCR product PCR
17 3. Estimate relationships based on extent of DNA similarity.At variable DNA positions, related groups will tend to share the same nucleotide. The sheer number of characters is helpful to distinguish the ‘phylogenetic signal’ from noise. ATGTTGGCAGTCCGATGTAAGC ATGTTGGCAGTCCGATGTAAGC ATGTTGGCAGTCCGATGTAACC ACGGTAGCAGTCTGATGTATCC ACGGTAGCAGTCTGATGTATCC ACGGTAGCAGTCTGATGTATCC CTGCTGGTAGTCGTTTGTAACC CTGCTGGTAGTCGTTTGTAACC CTGCTGGCAGTCGGTTGTAACC ATGCTGGCAGTCGGGTGTAACC Molecular phylogeny of taxa A-I. ATGGTGGCAGTCGGGTGTCACC Colored letters = different from top sequence (taxon G)
18 The elegant idea behind DNA sequencingTechnology changes quickly, but for many years we’ve used Sanger’s cool trick. In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators or dideoxynucleotides. This method is still in use today. What is the basis of Sanger’s method? Fred Sanger Shared with Walter Gilbert and Paul Berg
19 DNA Polymerase Action
20 Mechanism of DNA polymerization
21 Dideoxy Nucleotide
22 Dideoxies block elongationH ddGTP X
23 DNA Sequencing Reactions
24 Gel Electrophoretic Fractionation of Products
25 Fractionating DNA MoleculesDNA is negatively charged at neutral pH DNA will travel towards a positive charge DNA electrophoresed in a gel will travel according to its chain length
26 Detecting DNA Early experiments used radio-isotopically labeled DNA (primers) Now we use fluorescently labeled dideoxynucleotides
27 Perform cycle sequencing in PCR machinedenaturation Primer annealing Product extension Reaction steps seems a lot like PCR, but not quite…
28 Use of capillaries filled with a matrix for higher through-put of sequencing reactions.e.g. ABI 3730
29 In sum: From PCR to sequence data1. Purify PCR product 2. Set up sequencing reaction 3. Perform cycle sequencing 5. Read order of terminators (DNA sequence) 4. Resolve sequence fragments
30 Quality of sequence data may vary, depending on:Purity and concentration of template DNA Presence of extra PCR bands (artifacts) Quality of dye-terminators, electrophoresis matrix, and other reagents Ideally, look at chromatograms and convince yourself that base calls are robust.
31 DNA sequence assembly: Combining sequence reads to build the entire sequence of the template DNAPCR PRODUCT of 1,200 bp Sequence read #1 Sequence read #2 Sequence read #3 Sequence “reads” typically range from bp. Often the total DNA fragment is much longer. Regions of overlap among sequence reads: Let us piece together the linear “puzzle” of the template DNA sequence. Often confirm base calls and improve overall add data quality.
32 Major sequencing centers have facilities to sequence multiple full genomes each yearDue to genomic research over the past decade, DNA sequencing has become: Increasingly automated Higher-throughput Less $$ per reaction And (good news) more accessible to researchers and educators
33 Possible avenues for DNA sequencing:Many public universities have DNA sequencing facilities in house. Such facilities are usually able to run small numbers of reactions for a cost of ~$10-15 per reaction, but often much less for “internal users” with ties to the university. Ideal situation: Team up with faculty at your local university to act as facilitators. Even if DNA sequencing not an option for classroom labs, important to discuss: how DNA sequencing is performed, how data is evaluated, what information can be obtained from the data, what biologically important concepts can be learned, ethical issues surrounding its interpretation and use.
34 Ethical issues involving DNA sequencingTruro: A tranquil Cape community shaken by the murder of a young woman in January 2002
35 Should police have requested DNA of every man in Truro??
37 DNA sequencing Why? – Identifies Organisms How? – Dideoxy SequencingATGTTCTATCCCATTCATTTTGACGTTATTGTTGTTGGAGGAGGTCATGCTGGGACAGAAGCAGCTTTGGCTTCTGCTAGGATGCAATGTAATACGCTTTTGATTACTCATAATATTGATACTCTTGGACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGAAGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGATGAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCAAAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTTTCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCTTGTTATATTACCCATACTAATAACAAAACACATGAAATAGTTAGATCAAATTTATATCAGAGTCCTATGTATACAGGTTTAATAAAAGGAATAGCACCTCGGTATTGTCCATCTATAGAAGACAAAATAACTCGTTTTTCAGATCGTAATGCTCATCAAATTTTTTTAGAACCTGAAGGTTTGACAACACCTGAAGTATATCTTAATGGTATTTCTACTAGCTTACCTTTTTGTGTACAAATGCAAATGATTAAATCAATTCAGGGATTAGAGAATGCTTGTATAATTAGGCCAGGTTATGCTATTGAATACGATTTTTTTGATCCTCGTGATTTAAAATTAACATTAGAGAGTAAAATTATTTCTGGATTATTTTTTTCTGGTCAAATTAATGGTACTACTGGTTATGAAGAAGCAGCTGCCCAAGGATTATTGGCCGGAATTAATGCAGCTAGGTTTTCCAAAAATAAAGAAGGATGGTACCCTAGAAGAGATCAGGCATATTTAGGGGTGCTTGTAGATGATCTCTGTACACATGGAACAGAAGAACCTTATCGTATGTTTACTTCGCGTGCTGAATATCGTCTGTCTTTACGTGAGGATAATGCTGATTTAAGATTAACTGAAATCGCGCGACAGTTAGGTTTGATAGATGAGTCACGGTGGAAAGCTTTTTGTTGCAAAAAAGAAAACATTGAAAAAGAACGTCAGAGATTACGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAACACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAAGTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAATAAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTATAATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGACAAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTATCACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTAATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGGGTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAATAATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTATGTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAATAGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAAATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGTGTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGTGTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTCTATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACAACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTTGTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAATATTTTTTGAAAGGTGGTTTATGTCAGGAATCAAAGGCACTTCCCAAGAATATATTGGACATCATTTATATCATCTGCAATTTGATTTAAGTACTTTTTCGTTAGTGAGCTCGGAGAATACTTCTTCATTCTGGGTATTAAATGTAGATTCGATGTTTTTTTCAATATTATTGGCTACTTTATTTTTATTAATTTTCGGTCGTTTGGCTACAGTGGCAACTTATGCGGTTCCCACAAAACTGCAAGTGTTTATTGAGTTAGTAATATTGTTTATTGATAGCAATGTAAAAGATATGTTTCATGGTAAAAATAAACTAATTGCGCCATTATCTATGACGGTTTTTGTTTGGATTTTTTTAATGAATACCATGGATTTATTTCCCATAGATTTATTTCCTGCTATAGCTAAATTGTTAGGATTACCTGCTTTACGTGTTGTGCCGTCTGCCGATGTGAATATCACTTCTTCGCTAGCTTTAAATGTATTTGTACTTGTTATGTACTACAATATTTACGTTAATGGCGTTCATGGTTTTATTAAAGGACTGATGTATCATCCATTTAATCATCCAACATGTATTCCTATTAATTTTATTATTGAAGTTGTTAGTTTGTTATCTAAACCAGTATCACTTAGTCTTAGATTATTTGGCAATATGTATTCTGGTGAGTTGATTTTTATTTTGATATCTGGTTTATTACCATGGTGGGGACAATGGGTTTTAAATTTACCATGGGCTATTTTTCATATTTTAGTCGTTACATTACAGGCTTTTATTTTTATGGTTTTAACGGTGATTTATTTATCTACAGCCCATGACTCCTGTTAAAATGAATCATGTAATACAGAAGATTGCAGAGAGGTTATTATGGAACATTTAAATTTTGATATGTTATATATTGCTGCAGCAATAATGATGGGATTAGCAGCAATTGGAGCGGCTATCGGTATTGGCATCTTAGGTAGTAAATTTTTAGAGGGGGCTGCACGTCAACCAGATCTCATTCCGATCCTTCGAACTCAGTTTTTTATTGTTATGGGATTGGTTGATGCAATACCAATGATTACTGTAGGTCTTGGTTTGTATGTGATGTTTTCTGCGGTTTAACAATGAAAATTAATTGCATGTATTTATTATATTGATTATTAAATATCAGGATTTGCGCTGTGAATCTTAATGCAACAATATTAGGTCAAACTATTTCATTTGTTTTGTTTGTTTGGTTTTGTATGAAGTATGTGTGGTATCCATTTATATCTATTATTGAGAAACGCCAAAAAGAAATTTCTGATAATTTAGTTTCTGCTACTCATGCCAAAACAGAATCTGAGCGTGTCAATGCTGAAGCTTTGCTTTGTTTGAGACAGGCTCGGGTCAAAGCTCAAGAAATTATAAAACAAGCAAATAAATGTAAAATGCAAATAATTAATGAAGCTAAACATGAAGCTGAAAAGGAGCAAAGTAGAATTTTATCTCAAGCGCGAGAACAGATTATTTATGAAAGAAAACGTGTTACTGACGAATTAAGAAAGCAAATTAGCGAACTTGTAATTGAGGGTACAGAGAAAGTTATAGAACATTCTATAAATGAAATGATTGATATAGATCTTTTAAATAATATTATTAATACGTTGTCATATAAGGATTAGATGTCTAGTATGCTTGTGGTTGCGCGTACGTATGCTCAAGCTATATTTGATATAGCTGTAGAACAGAAAAATATAAACAAGTGGAAATCAGTTCTTGATTTATTTTCTGAGATTAGTCTAAATAGACTAGTACAATCTTTATTTTTTAGATGTTTAGAACCAAAAAGATTGTCAGATATATTTATTGCTATTTGTGAAGATTACCAAAAGAAACAAGTTGATACCTTCAGTAAAAATATAATCTATATTATGGCGGAAAATAATCGTTTATTATTATTACCAATTGTATTTAAAGAGTTTACTTATTTATGTTCTATATATGTTCATACTGTAGAAATAGAAATTATCTCTGCTTGGCCTTTGAAGTATAATCAGCTGAAAAAAATTACTGATATAATGGCTAAACGTTTATCTAAAACAGTGAATCCAGTACACAAAGTAGATAAAGATATATTGGCTGGTGTAATTATTCGTATTGGAGATACTGTGATTGATGGAAGTATACGTGGACGTATTTTTCGCTTAAATCACGTACTACAATCTTAATATTTTAACAGTAGTTAAAAGGTATAAACAAAAGATTATGCAATTAAATTCAAATGAAATTTCAGATAATGGAGAAGTTATTATTAATGATTTAAAGTTATTTTATAATAAAGCTAGACAAACTAAAATTACAGAAGAACTTACAGAAATTGTTTCAGGAGCTTCTGTAATATAAACTTAAAAGATAGTTAGAGGTATAATTAATATGAGTTCTGGAAAAATTGTCCAGGTTATTGGAGCGGTGGTTGATGTTGCGTTCAATCAAGATGTGGTACCGACTGTATACCATGCACTTGAGGTGTAATACTTATATTTTCCCGTATAGCTCAGATGTTGCACAATTAAATAATTTTCTTAAAACACCTTTAACACATGAAACAAATGGCGAAGATCTTTTGAGAAGGCCAGAAATTAATTATAAAAAGTTATCTCAACTAAGTACTTTTAGTCCATCTATATTGGATCGTCAAGTATTCGAGCAAATTGAAATTCAAATAAAATATGAAGGTTATATTCGTCATCAACAAGAAGAAATAAAGAGACATATTTATAATGAAAATACATTGTTACCGACTGATATAGATTTCAATATTGTTTCTGGATTATCTCAGGAGGTCATTGACAAACTCAATAATTATAAACCTTATTCTATTGGGCAGGCTTCTCGCATTTCTGGTATCACTCCTGCGGCTATTTCTAATTTATTGGTTTGGTTAAAAAAACAAGGTTTGTTAGAGCATAATACATGCTAATCTGTTGAATAATGTATTTTCTATTAATCAAGGTATTATTTAATTTCCTATATAGGGTGTTTGTGATTGTTACAATATGGAGAGAGTTGATCGTGTAGAAAACCGGTATATTTATGTCTTTTCCAATAATAATTTTATAGAAATAATAATAGTAATTAGATTATTATTTTAATGGGTTTGTTATGTATGGCATTATTTTTAATATTATACGGTTGTTTATACGTATCTAAATTTGCGCTTTGAATTTTTTTGAAAATAGGATTATACTGAACTTTTAGTTGTATACAAGTGTATTATTTCATATTTTTGAATAAATATACGTGCTGTACTAATGGTTATTAATTACAAGATAAATTACTTGTCGACCGAACTATAGTTCGGTCGTGTGTTTGTTTCTGTAAGTTGTAACATATTATGTGAAATTTACTAATTAATTCATAGTGTCATTTTAATTCTCTTTTATTGCATTAGTATATGGTTGTGTAAAAAGAGAGAGAGTGTTCTTGTTATTTTCTATTAGTACTAATTTTTTAAAAGTTGATGCAGGCACTGTCGTTCTTTTCGGATACAACATCATTGTAAGTTATATATAAAAATGATTTAGAATTTCGACATGTTAGTGACAAGTTGTATATCGAGGTTGTAATGTTGTATTATACAAGAACATTGTAAAATATTTATAGTATATTGCAGCCAATATTTTTTGAAAGGTGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCAAGCGTCAGTAGAAGATTTAATAGTTAGTGGGAACAAGATTGTTGGAGTAATTACTCCAAAATTAGGAATGAAATTTAGTGGTACGTCTGTTGTGTTGACAACCGGAACCTTTCTCAATGGTAAGATTCATATTGGGATGAATAATTTTAGAGGAGGTCGATCTGGAGATTCGGAGTCATCGTCATTGTTATCAGAGCGATTGAAAGAATTGTCTTTTCAGATTAGTCGGTTAAAAACGGGTACTTCTCCTCGTGTGCATACCAAAGGGATAAATTTTGGTTCTTTACGTGCACAATATAGTGATGATCCCATTCCTGTGTTTTCATTTATAGGATCTACAAAACTACATCCTACGCAGGTGCCT DNA sequencing Why? – Identifies Organisms How? – Dideoxy Sequencing Introduces Biological Machines Where? – Universities, MBL Societal Impact? – Forensics, Insurance DNA detective
38 DNA-based Technologies Lecture - Dr. Seth BordensteinPCR Animation
39 Dideoxies block elongationH ddGTP X
40 Illumina Next Gen Sequencing
41 Fascinating. Now how do we actually sequence ??