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Sydney Brenner et al. Jan 26, 2007

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1 Sydney Brenner et al. Jan 26, 2007
Gene expressions analysis by massively parallel signature sequencing (MPSS) on microbead arrays Sydney Brenner et al. Jan 26, 2007

2 DNA-basic definitions
Nucleotide (Base) - consists of a sugar, phosphate and a base Base-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.

3 DNA Sequencing Sequencing: determining the nucleotide order of a given DNA fragment, called the DNA sequence. Sanger Method (’75): Chain termination Prepare 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 I A lot more deoxynucleotides than dideoxynucleotides Sort based on length (gel) Animation:

4 Restriction Enzymes 1978 Nobel Prize in medicine (awarded to Werner
Arber, Daniel Nathans, and Hamilton Smith) Enzymes that cut double stranded DNA The cleaved chemical bonds can be reformed by ligases Restriction enzyme cuts only double-helical segments that 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 distances II: cleavage occurs at specific sites at or close to the recognition sequence Restriction: 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.

5 Fluorescence Activated Cell Sorting (FACS)
First cell sorter: Mack Fulwyler (1965) Expanded by Len Herzenberg Cells 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 cells Electrical charge is used to sort the cells

6 Motivation Goals (upon determination of human genome):
To discover and understand the function and variation of genes How do these qualities affect health and disease? Available techniques: Hybridization of probes into microarrays Advantages: 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 differences Counting of tags or signatures of DNA fragments Advantages: Statistically more robust, don’t require standardization or repetition, precision and accuracy can be increased by increasing the size of the sample Disadvantages: Difficult to realize routinely and not cost effective

7 Massively Parallel Signature Sequencing (MPSS)
Cloning on microbeads 1% of tags # of transcripts: e4 # of oligonucleotides: 1.67e7 # of conjugates: 5-7e11 Oligonucleotides: kind of like matching locks………short pieces of DNA that readily combine with their complements…..32mer in this example We want to make sure that # of tags is at least a 100 times the number of templates this will ensure that if we take 1% of tags, we have a sample where all DNA’s are represented and they all have unique tags with at least 99% probability.

8 Massively 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 molecule Micobeads have the complementary tags for all tags but since we took 1% of tags….1% of microbeads are filled

9 Massively Parallel Signature Sequencing (MPSS)

10 Decoding… 1 TNNN…Fx1 NTNN…Fx2 NNCN…Fx3 NNNC…Fx4 TTCC 43 x 16 3 2
Ligate 1024 encoders 43 x 16 Decoders for Fx1…4 3 2 16x decoding per cycle….repeat

11 Massively Parallel Signature Sequencing (MPSS)

12 Massively Parallel Signature Sequencing (MPSS)

13 Comparison to conventional methods
Compared to PE Biosystems, model 377 DNA Sequencer

14 Discussion Method does not require separation of fragments to generate sequence information Time 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 bases MPSS: analyze millions of templates to give sequences of length few 10s of bases

15 Critique Summary Strong points: A powerful and innovative technique
Lots of work done to prove the functionality Weak points Not the clearest paper (too much information compacted in a few sentences) There are more efficient ways of encoding/decoding the adaptors The figures are better shown in a different order It would be good to include some more information about the cloning of the microbeads I could use more comments on how good the results are (is it just me!?)

16 Better encoding (Hansen’s method)
1 2 3 4 5 6 7 A T C G . AAAT AAAG ,7 23 = 8 different encoders required as opposed to 16 used here.

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