1. Bruce Blumberg (blumberg@uci.edu)
BioSci D145 Lecture #3
Bruce Blumberg
4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment)
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TA – Riann Egusquiza
4311 Nat Sci 2– office hours W 9:30-11:30
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check regularly for announcements, etc..
Updated lectures will be posted on web pages after lecture
Don’t forget to discuss term paper topics with me
Last year’s midterm is now posted
BioSci D145 lecture 1 page 1 ©copyright Bruce Blumberg All rights reserved

2. Why should any funding agency give you money to pursue this research?
Term paper outline
Title of your proposal
A paragraph introducing your topic and explaining why it is important; i.e., what impact will the knowledge gained have.
Why should any funding agency give you money to pursue this research?
NIH now requires a statement of human health relevance for all grant applications
NSF wants to know what is the intellectual merit of your proposed research and what broader impacts of your proposed research
Present your hypothesis
A supposition or conjecture put forth to account for known facts; esp. in the sciences, a provisional supposition from which to draw conclusions that shall be in accordance with known facts, and which serves as a starting-point for further investigation by which it may be proved or disproved and the true theory arrived at.
Enumerate 2-3 specific aims in the form of questions that test your hypothesis
At least one of these aims needs to have a strong “whole genome” component
This is not a review article – propose something new.
BioSci D145 lecture 4 page 2 ©copyright Bruce Blumberg All rights reserved

3. DNA sequencing = determining the nucleotide sequence of DNA
DNA sequence analysis
DNA sequencing = determining the nucleotide sequence of DNA
Originally two main methods
shared Nobel prize in 1980
Chemical cleavage – Maxam and Gilbert
Enzymatic sequencing (based on polymerization reaction)
Nobel Prize in Chemistry 1980
Walter Gilbert (Harvard) & Frederick Sanger (MRC Labs)
(Sanger also won Nobel in 1958 for protein sequencing)
BioSci D145 lecture 4 page 3 ©copyright Bruce Blumberg All rights reserved

4. One of the first reasonable sequencing methods
DNA sequence analysis
Maxam and Gilbert
One of the first reasonable sequencing methods
Very popular in late 70s and early 80s
VERY TEDIOUS!!
Totally superceded by dideoxy sequencing now
BioSci D145 lecture 4 page 4 ©copyright Bruce Blumberg All rights reserved

5. DNA sequence analysis (contd)
Dideoxy sequencing – Sanger 1977
Virtually all routine sequencing is done this way now
Requires modified nucleotide
2’3’-dideoxy dNTP
DNA polymerase incorporates the ddNTP and chain elongation terminates
Original method used 4 separate elongation reactions
Products separated by denaturing PAGE and visualized by autoradiography
BioSci D145 lecture 4 page 5 ©copyright Bruce Blumberg All rights reserved

6. DNA sequence analysis (contd)
Dideoxy sequencing (contd) – Sanger 1977
Dideoxy NTPs present at ~1% of [dNTP]
Each reaction has identified end
In principle, all possible chain lengths are represented
varies by [dNTPs], [ddNTPs], [primer] and [template] and ratios
BioSci D145 lecture 4 page 6 ©copyright Bruce Blumberg All rights reserved

7. DNA sequence analysis (contd)
A C G T A C G T
A C G T
BioSci D145 lecture 4 page 7 ©copyright Bruce Blumberg All rights reserved

8. Automated DNA sequence analysis
How to improve throughput of sequencing?
Incorporate fluorescent ddNTPs, separate products by PAGE
Base calling and lane calling issues
Key advance was capillary sequencers
Separate DNA in a thin capillary instead of gel
Very accurate, no tracking errors, much more automation friendly
Trace files (dye signals) are analyzed and bases called to create chromatograms.
Chromatograms from opposite strands are reconciled with software to create double-stranded sequence data.
BioSci D145 lecture 4 page 8 ©copyright Bruce Blumberg All rights reserved

9. Automated DNA sequence analysis
Capillaries vs gels
Capillaries much faster – higher field strength possible
Fully automated = higher throughput
BioSci D145 lecture 4 page 9 ©copyright Bruce Blumberg All rights reserved

10. Applied Biosystems PRISM 377 (Gel, 34-96 lanes)
(Capillary, 96 capillaries)
BioSci D145 lecture 4 page 10 ©copyright Bruce Blumberg All rights reserved

11. PCR – polymerase chain reaction amplification of DNA
PCR is most routinely used method to amplify DNA
Exponential amplification of DNA by polymerases – Saiki et al, 1985
2n fold amplification, n= # cycles
35 cycles = 235 = 3.4 x 1010 fold
Originally used DNA polymerase I
Needed to add fresh enzyme at every cycle because heat denaturation of template killed the enzyme
Not widely used – too painful to do manually
Nobel Prize to Kary Mullis in 1993 for deciding to use Taq DNA polymerase for PCR
He was middle author on paper!
BioSci D145 lecture 4 page 11 ©copyright Bruce Blumberg All rights reserved

12. PCR – polymerase chain reaction amplification of DNA (contd)
Hot water bacteria:
Thermus aquaticus
Taq DNA polymerase
Life at High Temperatures by Thomas D. Brock
Biotechnology in Yellowstone
© 1994 Yellowstone Association for Natural Science
BioSci D145 lecture 4 page 12 ©copyright Bruce Blumberg All rights reserved

13. Cycle sequencing – fusion of PCR and fluorescent ddNTP sequencing
Combine PCR amplification with dideoxy sequencing – cycle sequencing
Linear amplification of template in the presence of fluorescent ddNTPs
When nucleotides are used up reaction is over
Separate on capillary electrophoresis instrument
Advantages
Fast, single tube reaction
Works with small amounts of starting material
Disadvantages
Still need to prepare high quality template to sequence
Cost and time
Many sequencing centers spend time, $$ on template prep
Automation requirements
BioSci D145 lecture 4 page 13 ©copyright Bruce Blumberg All rights reserved

14. Isothermal amplification – the solution to template preparation
How to make template preparation faster, easier and more reliable?
Eliminate automation requirement, amplify starting material in some other way
Φ29 DNA polymerase (aka TempliPhi)
Enzyme has high processivity and strand displacement activity
Isothermal reaction produces huge quantities of DNA from tiny amount of input
More efficient than PCR (no temp change, no machine, no cleanup)
BioSci D145 lecture 4 page 14 ©copyright Bruce Blumberg All rights reserved

15. Modern DNA sequence analysis
Cycle sequencing
Virtually all routine DNA sequencing today is done by cycle sequencing with fluorescent ddNTPs
ABI Big Dye chemistry
Template preparation still tedious for small scale
TempliPHi used in genome centers (no need for most automation)
Capillary sequencers predominant for small scale sequencing
Retrogen and similar companies
But, next generation sequencing has already rapidly displaced old technology in genome centers.
454 sequencing (Roche)
Solexa (Illumina) *dominant player at the moment*
SoLID (Applied Biosystems) (dead technology due to poor support)
3rd generation sequencing (individual DNA molecule) now available
e.g., Pacific Biosciences (sequence reads of 1,000-10K bases)
Oxford Biosciences Nanopore (read length 5 kb—200 kb)
BioSci D145 lecture 4 page 15 ©copyright Bruce Blumberg All rights reserved

16. Landmarks in DNA sequencing
DNA sequence analysis
Landmarks in DNA sequencing
Sanger, Nicklen and Coulson. Sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. 74, (1977).
Sanger, F. et al. The nucleotide sequence of bacteriophage ΦX174. J Mol Biol 125, (1978).
Sutcliffe, J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol 43, (1979).
Sanger et al., Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol 162, (1982).
Messing, J., Crea, R. & Seeburg, P. H. A system for shotgun DNA sequencing. Nucl.Acids Res 9, (1981).
Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, (1981).
Deininger, P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem 129, (1983).
Baer et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310, (1984). (189 kb)
Innis et al. DNA sequencing with Taq DNA polymerase and direct sequencing of PCR-amplified DNA Proc. Natl. Acad. Sci. 85, (1988)
BioSci D145 lecture 4 page 16 ©copyright Bruce Blumberg All rights reserved

17. DNA sequence analysis (contd)
Landmarks in DNA sequencing (contd).
Haemophilus influenzae (1.83 Mb)
Mycoplasma genitalium (0.58 Mb)
Saccharomyces cerevisiae genome (13 Mb)
Methanococcus jannaschii (1.66 Mb)
Escherichia coli (4.6 Mb)
Bacillus subtilis (4.2 Mb)
Borrelia burgdorferi (1.44 Mb)
Archaeoglobus fulgidus (2.18 Mb)
Helicobacter pylori (1.66 Mb)
first bacterium sequenced, human pathogen
smallest free living organism
first Archaebacterium
Lyme disease
first sulfur metabolizing bacterium
first bacterium proven to cause cancer
BioSci D145 lecture 4 page 17 ©copyright Bruce Blumberg All rights reserved

18. DNA sequence analysis (contd)
Landmarks in DNA sequencing (contd)
Treponema pallidum (1.14 Mb)
Caenorhabditis elegans genome (97 Mb)
Deinococcus radiodurans (3.28 Mb)
Drosophila melanogaster (120 Mb)
Arabidopsis thaliana (115 Mb)
Escherichia coli O157:H7 (4.1 Mb)
2001 – draft Human “genome”
2002 – mouse genome
2002 – Ciona intestinalis
2003 – “complete “human genome
2004 – rat genome
2006 – Human “genome” complete sequence of all chromosomes
Many more genomes underway, check JGI, Sanger and other web sites
resistant to radiation, starvation, ox stress
Pathogenic variant of E. coli
Primitive chordate
BioSci D145 lecture 4 page 18 ©copyright Bruce Blumberg All rights reserved

19. Complete DNA sequence (all nts both strands, no gaps)
DNA Sequence analysis
Complete DNA sequence (all nts both strands, no gaps)
complete sequence is desirable but takes time
how long depends on size and strategy employed
which strategy to use depends on various factors
how large is the clone?
cDNA
genomic
How fast is sequence required?
sequencing strategies
primer walking
cloning and sequencing of restriction fragments
progressive deletions
Bidirectional, unidirectional
Shotgun sequencing
whole genome
with mapping
map first (C. elegans)
map as you go (many)
BioSci D145 lecture 4 page 19 ©copyright Bruce Blumberg All rights reserved

20. DNA Sequence analysis (contd)
Primer walking - walk from the ends with oligonucleotides
sequence, back up ~50 nt from end, make a primer and continue
Why back up?
Need to see overlap to be sure about sequence you are reading
BioSci D145 lecture 4 page 20 ©copyright Bruce Blumberg All rights reserved

21. DNA Sequence analysis (contd)
Primer walking (contd)
advantages
very simple
no possibility to lose bits of DNA
restriction mapping
deletion methods
no restriction map needed
best choice for short DNA
disadvantages
slowest method
about a week between sequencing runs
oligos are not free (and not reusable)
not feasible for large sequences
applications
cDNA sequencing when time is not critical
targeted sequencing
verification
closing gaps in sequences
BioSci D145 lecture 4 page 21 ©copyright Bruce Blumberg All rights reserved

22. DNA Sequence analysis (contd)
Cloning and sequencing of restriction fragments
once the most popular method
make a restriction map, subclone fragments
sequence
advantages
straightforward
directed approach
can go quickly
cloned fragments often useful otherwise
RNase protection, nuclease mapping, in situ hybridization
disadvantages
possible to lose small fragments
must run high quality analytical gels
depends on quality of restriction map
mistaken mapping -> wrong sequence
restriction site availability
applications
sequencing small cDNAs
isolating regions to close gaps
BioSci D145 lecture 4 page 22 ©copyright Bruce Blumberg All rights reserved

23. DNA Sequence analysis (contd)
nested deletion strategies - sequential deletions from one end of the clone
cut, close and sequence
Approach
make restriction map
use enzymes that cut in polylinker and insert
Religate, sequence from end with restriction site
repeat until finished, filling in gaps with oligos
advantages
Fast, simple, efficient
disadvantages
limited by restriction site availability in vector and insert
need to make a restriction map
BioSci D145 lecture 4 page 23 ©copyright Bruce Blumberg All rights reserved

24. DNA Sequence analysis (contd)
nested deletion strategies (contd)
Exonuclease III-mediated deletion
cut with polylinker enzyme
protect ends -
3’ overhang
phosphorothioate
cut with enzyme between first cut and the insert
can’t leave 3’ overhang
timed digestions with Exonuclease III
stop reactions, blunt ends
ligate and size select recombinants
sequence
advantages
unidirectional
processivity of enzyme gives nested deletions
BioSci D145 lecture 4 page 24 ©copyright Bruce Blumberg All rights reserved

25. DNA Sequence analysis (contd)
Nested deletion strategies
Exonuclease III-mediated deletion (contd)
disadvantages
need two unique restriction sites flanking insert on each side
best used successively to get > 10kb total deletions
may not get complete overlaps of sequences
fill in with restriction fragments or oligos
applications
method of choice for moderate size sequencing projects
cDNAs
genomic clones
good for closing larger gaps
Small-scale sequence analysis – how is it practiced today?
Primer walking
ExoIII-mediated deletion with primer walking
BioSci D145 lecture 4 page 25 ©copyright Bruce Blumberg All rights reserved

26. Genome sizes for most eukaryotes are large (108-109 bp)
Genome sequencing
The problem
Genome sizes for most eukaryotes are large ( bp)
High quality sequences only about bp per run (Sanger)
Nextgen sequences ~150 bp/read
The solution
Break genome into lots of bits and sequence them all
Reassemble with computer
The benefit
Rapid increase in information about genome size, gene comparisons, etc
The cost
3 x 109 bp(human haploid genome) ÷ 600 bp/reaction = 5 x 106 reactions for 1x coverage!
Need both strands (x2), need overlaps and need to be sure of sequences
~ reactions/runs required for a human-sized genome
About $1-2 per reaction these days, ~$8 commercially.
BioSci D145 lecture 4 page 26 ©copyright Bruce Blumberg All rights reserved

27. Genome sequencing (contd)
Shotgun sequencing NOT invented by Craig Venter
Messing 1981 first description of shotgun sequencing
Sanger lab developed current methods in 1983
approach
blast genome into small chunks
clone these chunks
3-5 kb, 8 kb plasmid
40 kb fosmid jump repetitive sequences
sequence + assemble by computer
A priori difficulties
how to get nice uniform distribution
how to assemble fragments
what to do about repeats?
How to minimize sequence redundancy?
BioSci D145 lecture 4 page 27 ©copyright Bruce Blumberg All rights reserved

28. Genome sequencing(contd)
BioSci D145 lecture 4 page 28 ©copyright Bruce Blumberg All rights reserved

29. Genome sequencing(contd)
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30. Genome sequencing (contd)
Shotgun sequencing (contd)
How to minimize sequence redundancy?
Best way to minimize redundancy is map before you start
C. elegans was done this way - when the sequence was finished, it was FINISHED
mapping took almost 10 years
mapping much too tedious and nonprofitable for Celera
who cares about redundancy, let’s sequence and make $$
There is scientific value to draft genomes, too.
why does redundancy matter?
Finished sequence today costs about $0.50/base
Note that 10x, % coverage leaves at least 150 kb unsequenced
BioSci D145 lecture 4 page 30 ©copyright Bruce Blumberg All rights reserved

31. Other sequencing technologies
Sequencing by hybridization
Construct a high-density microchip with all possible combinations of a short oligonucleotide
Up to 25-mers
By photolithography
Synthesized on chip directly
Label and hybridize fragment to be sequenced
Wash stringently
Read fluorescent spots
Reconstruct sequence by computer
BioSci D145 lecture 5 page 31 ©copyright Bruce Blumberg All rights reserved

32. Other sequencing technologies (contd) stoopped here)
Sequencing by hybridization rarely used for de novo sequencing
Extremely fast and useful to sequence something you already know the sequence of but want to identify mutation - resequencing
Disease causing changes
e.g in mitochondrial DNA
SNP discovery
Works best for examining sequence of <10 kb
BioSci D145 lecture 5 page 32 ©copyright Bruce Blumberg All rights reserved

33. Other sequencing technologies (contd)
SNP discovery
Photo shows mitochondrial chip
Right panel shows pairs of normal (top) vs disease (bottom) (Leber’s Hereditary Optic Neuropathy)
Top 3 disease mutations
Bottom control with no change
BioSci D145 lecture 5 page 33 ©copyright Bruce Blumberg All rights reserved

34. Other sequencing technologies – Next Generation sequencing
2nd generation = high throughput, short sequences
3rd generation = single molecule sequencing
Small number of sequence templates (thousands) but very long reads (~105 bp)
What is the immediate implication of this technology for genome assembly?
See Metzger, M.L. (2010) Sequencing technologies — the next generation, Nature Reviews Genetics 11,
We should now be able to completely sequence large insert clones directly and avoid fragmentation by repetitive elements!
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