Presentation on theme: "Sydney Brenner et al. Jan 26, 2007"— Presentation transcript:
1Sydney Brenner et al. Jan 26, 2007 Gene expressions analysis by massively parallel signature sequencing (MPSS) on microbead arraysSydney Brenner et al.Jan 26, 2007
2DNA-basic definitions Nucleotide (Base) - consists of a sugar, phosphate and a baseBase-Pair Rule – Hydrogen bonds (A-T, C-G)Gene - a segment of DNA that codes for a protein, which in turn codes for a trait (skin tone, eye color,…etc), a gene is a stretch of DNA.DNA Polymerase - Enzymes that catalyze the polymerization of deoxyribonucleotides alongside a DNA strand, which they "read" and use as a template. The newly-polymerized molecule is complementary to the template strand and identical to the template's partner strand.
3DNA SequencingSequencing: determining the nucleotide order of a given DNA fragment, called the DNA sequence.Sanger Method (’75): Chain terminationPrepare single strand DNA (heating)Add a mixture of deoxy nucleotides (dATP,dGTP, dCTP, dTTP)Add a mixture of dideoxy nucleotides(ddATP,ddGTP,ddCTP, ddTTP)Add DNA polymerase IA lot more deoxynucleotides than dideoxynucleotidesSort based on length (gel)Animation:
4Restriction Enzymes 1978 Nobel Prize in medicine (awarded to Werner Arber, Daniel Nathans, and Hamilton Smith)Enzymes that cut double stranded DNAThe cleaved chemical bonds can be reformed byligasesRestriction enzyme cuts only double-helical segmentsthat contain a particular nucleotide sequence (i.e.recognition sequence)Types of Restriction enzymes: I, II, III:I,III: recognize specific sequences but the cleavage sites are at variable distancesII: cleavage occurs at specific sites at or close to the recognition sequenceRestriction: R.E’s were discovered in E coli….they appeared to be restricting the infection by certain bacteriophages. First practical application: manipulating E coli bacteria to produce insulin for diabatics.Type II enzymes are further classified according to their recognition site. Most type II enzymes cut palindromic DNA sequences, while type IIa enzymes recognise non-palindromic sequences and cleaveage outside of the recognition site, and type IIb ones cut sequences twice at both sites outside the recognition sequence.
5Fluorescence Activated Cell Sorting (FACS) First cell sorter: Mack Fulwyler (1965)Expanded by Len HerzenbergCells are tagged by antibodies linked to fluorescent dye. The antibody is bound to a protein that is uniquely expressed in the cells that we want sorted.The nozzle vibrates to form drops which contain single cellsElectrical charge is used to sort the cells
6Motivation Goals (upon determination of human genome): To discover and understand the function and variation of genesHow do these qualities affect health and disease?Available techniques:Hybridization of probes into microarraysAdvantages: large scale, capable of detecting a wide range of gene expression levels.Disadvantages: variability due to probe hybridization, cross reactivity, element to element differences, and microarray to microarray differencesCounting of tags or signatures of DNA fragmentsAdvantages: Statistically more robust, don’t require standardization or repetition, precision and accuracy can be increased by increasing the size of the sampleDisadvantages: Difficult to realize routinely and not cost effective
7Massively Parallel Signature Sequencing (MPSS) Cloning on microbeads1% of tags# of transcripts: e4# of oligonucleotides: 1.67e7# of conjugates: 5-7e11Oligonucleotides: kind of like matching locks………short pieces of DNA that readily combine with their complements…..32mer in this exampleWe want to make sure that # of tags is at least a 100 times the number of templatesthis will ensure that if we take 1% of tags, we have a sample where all DNA’s arerepresented and they all have unique tags with at least 99% probability.
8Massively Parallel Signature Sequencing (MPSS) PCR is used to amplify the sample. The resulting single stranded DNA’s are hybridized with a population of microbeads. (note: 1% of microbeads are loaded)Separate loaded microbeads from unloaded ones using FACS.Each microbead has a population of identical copies of a single kind of template moleculeMicobeads have the complementary tags for all tags but since we took 1% of tags….1% of microbeads are filled
13Comparison to conventional methods Compared to PE Biosystems, model 377 DNA Sequencer
14DiscussionMethod does not require separation of fragments to generate sequence informationTime series of spatially localized microbeads (can pack beads closely in monolayers)The main advantage: parallel nature of the process-millions of templates can be handled together without need for separation. (Ideal for gene expression)Conventional sequencing: analyze thousands of templates to give sequences with 100s of basesMPSS: analyze millions of templates to give sequences of length few 10s of bases
15Critique Summary Strong points: A powerful and innovative technique Lots of work done to prove the functionalityWeak pointsNot the clearest paper (too much information compacted in a few sentences)There are more efficient ways of encoding/decoding the adaptorsThe figures are better shown in a different orderIt would be good to include some more information about the cloning of the microbeadsI could use more comments on how good the results are (is it just me!?)
16Better encoding (Hansen’s method) 1234567ATCG.AAATAAAG ,723 = 8 different encoders required as opposed to 16 used here.