Lecture 8: Sequencing and SNP. Sept 15, 2006

Transcription

1 Lecture 8: Sequencing and SNP Sept 15, 2006

2 Announcements Random questioning during literature discussion sessions starts next week for real! Schedule changes Moved QTL lecture up Removed landscape genetics lecture Added a review session before Thanksgiving Added term paper page Do the reading! Check website regularly!

3 Last Time AFLP Sequence-tagged markers: SCARs Microsatellites

4 Today DNA labeling Quick screening methods for detecting sequence variation Sequencing Methods Direct versus cloning SNP

5 DNA labeling options Post-staining ethidium bromide, sybr green silver staining End-labeling versus labeling of dntp for incorporation End-labeling: one strand only, uniform signal dntp labeling: both strands, signal proportional to length (usually) Options: Radioactive isotopes: usually 35 S, 33 P, 32 P Nonradioactive conjugates: Streptavidinbiotin-antibody, Digoxigenin Fluorophores 30/HTML/Kothe_html_2fd5c98d.png

6 Fluorophores Can be used for end-labeling or dntp labeling Characteristic excitation and emission for each dye Key is to choose lasers that optimally excite different dyes, and detection filters that optimally differentiate them terbasics_spectrum.gif h/2006/ /fig2.png

7 Single-Strand Conformational Polymorphism 1. Amplify Target Sequence 2. Denature product with heat and formamide 3. Analyze on native (nondenaturing) polyacrylamide gel or capillary 4. Base sequence determines 3-dimensional conformation, and rate of migration: can detect single base changes

8 Denaturing Gradient Gel Electrophoresis 1. Amplify Target Sequence 2. Run product on gel with denaturing gradient (parallel or perpendicular to direction gel runs) 3. Product begins denaturing at a certain point, depending on base sequence: greatly retards migration and allows discrimination of alleles based on small sequence differences 4. Denaturing gradient gels are difficult to produce: use perpendicular gradient to identify optimal conditions, move to CDGE: constant denaturant gel electrophoresis

9 Cleaved Amplified Polymorphic Sequence 1. Amplify Target Sequence 2. Cut with a restriction enzyme that differentiates alleles X Allele 1 Allele 2 3. Alleles can be differentiated by size based on loss or gain of restriction site; May be able to analyze on agarose gel Fairly simple analysis (cutting can be a hassle) Requires sequence information from several alleles (or luck)

10 DNA Sequencing Sanger method was big breakthrough (1977) Relies on chain termination at specific bases by using dideoxynucleotides in low concentration Random termination of chain when ddntp is incorporated Lodish et al. 2004

11 BigDye Terminator Sequencing With primer labeling, must do 4 separate reactions Labeling ddntp with four different fluorescent dyes allows multiplexing in one reaction, analyzing in one lane or capillary BigDye optimized for best signal Quality scores empirically determined: Q=-10log 10 (p), where p is probability of error. e.g., if p=1x10-4, Q=40

12 Capillary Sequencing Revolutionized DNA sequencing: no more pouring and loading gels! Everything is done in multiples of 96 to take advantage of microplate format (12 x 8) Capillary sequencers can handle 1536 sequences at a time Automated data collection

13 Cloning and Sequencing of PCR Products Amplify specific gene product Clone into plasmid: TA plasmids take advantage of extra base added by Taq polymerase Sequence using universal primer Save money on primers Consistent sequencing results Sequences only one allele: simpler scoring Haplotypes

14 Direct Sequencing of PCR Products Direct sequencing bypasses cloning, thus streamlining the process Need unique PCR product Must remove primers and dntps Spin columns Gel purification Use exonuclease-1 to remove primer and shrimp alkaline phosphatase to deactivate dntps Both alleles sequenced simultaneously Complication is identification of heterozygotes

15 Shotgun Sequencing and Assembly Randomly clone pieces of sheared DNA Sequence ends Reassemble based on homology and paired ends

16 Pyrosequencing Release of pyrophosphate from nucleotides provides substrate ATP-sulfurylase Creates ATP ATP provides energy for luciferase Produces burst of fluorescence Treat with Apyrase to remove nucleotides Special machines add dntp at key steps Only 15 base read length usually

17 454 Sequencing New bead-based sequencing technology from 454 Life Sciences Combines pyrosequencing and bead-based genotyping Single run of machine can produce 25 Mb of 100 nucleotide sequences Tremendous potential for SNP discovery and genotyping on whole-genome scale

18 Sequencing Advantages Sequencing Disadvantages Highest resolution and most informative approach All levels of mutation accessible Expensive and slow Error rates can be high Clear mutation mechanism Ease of comparison across all organisms

19 SNPs A Single Nucleotide Polymorphism (SNP) is a single base mutation in DNA. The most common source of genetic polymorphism (e.g., 90% of all human DNA polymorphisms). Identify SNP by screening a sample of individuals from study population: usually 16 to 48 Ascertainment Bias: rare SNP go undiscovered Elevated H E for polymorphic loci Depressed H E on genome scale F ST depressed Once identified, SNP are assayed in populations using high-throughput methods