BioSci 203 lecture 22 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 22 - cDNA library screening &sequence characterization

BioSci 203 lecture 22 page 1 ©copyright Bruce Blumberg 2001. All rights reserved

Bio Sci 203 Lecture 22 - cDNA library screening &sequence characterization

• Bruce Blumberg ([email protected])

– office - 4203 McGaugh Hall

– 824-8573

– lab 5427 (x46873), 5305 (x43116)

– office hours Wednesday 1-2.

• Today

– Characterization of Selected DNA Sequences

• DNA sequence analysis


Analysis of genes and cDNAs

• Characterization of cloned DNA

– what things do we want to know about a new gene?

• Complete DNA sequence

– cDNA sequence

– genomic sequence?

– Restriction enzyme maps?

• Where are introns and exons?

– Particularly if knockouts are coming

• where is the promoter(s)?

– Alternative promoter use?

– Mapping transcription start(s)

• where and when is mRNA expressed?

– How abundantly is it expressed in each place?

– Is there any association between expression levels and putative function?

• What is the function of this gene?

– Loss-of-function analysis decisive

» knockout

» antisense

» mutant mRNA e.g. dominant negative

– gain of function may be helpful

» transgenic

» mutant mRNA - constitutively active transcription factor


Analysis of genes and cDNAs (contd)

• Landmarks in DNA sequencing

– Sanger, Nicklen and Coulson. Sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. 74, 5463-5467 (1977).

– Sanger, F. et al. The nucleotide sequence of bacteriophage ΦX174. J Mol Biol 125, 225-46. (1978).

– Sutcliffe, J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol 43, 77-90. (1979).

– Sanger et al., Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol 162, 729-73. (1982).

– Messing, J., Crea, R. & Seeburg, P. H. A system for shotgun DNA sequencing. Nucl.Acids Res 9, 309-21 (1981).

– Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, 457-65 (1981).

– Deininger, P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem 129, 216-23. (1983).

– Baer et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310, 207-11. (1984). (189 kb)

– Innis et al. DNA sequencing with Taq DNA polymerase and direct sequencing of PCR-amplified DNA Proc. Natl. Acad. Sci. 85, 9436-9440 (1988)


Analysis of genes and cDNAs (contd)

• Landmarks in DNA sequencing (contd).– 1995 - Haemophilus influenzae (1.83 Mb)

– 1995 - Mycoplasma genitalium (0.58 Mb)

– 1996 - Saccharomyces cerevisiae genome (13 Mb)– 1996 - Methanococcus jannaschii (1.66 Mb)

– 1997 - Escherichia coli (4.6 Mb)– 1997 - Bacillus subtilis (4.2 Mb)– 1997 - Borrelia burgdorferi (1.44 Mb)

– 1997 - Archaeoglobus fulgidus (2.18 Mb)

– 1997 - Helicobacter pylori (1.66 Mb)

– 1998 - Treponema pallidum (1.14 Mb)– 1998 - Caenorhabditis elegans genome (97 Mb)– 1999 - Deinococcus radiodurans (3.28 Mb)

– 2000 - Drosophila melanogaster (120 Mb)– 2000 - Arabidopsis thaliana (115 Mb)– 2001 - Escherichia coli O157:H7 (4.1 Mb)– 2001 - Human “genome”– 2002 – mouse genome– 2002 – Ciona intestinalis

• first bacterium sequenced, human pathogen

• smallest free living organism

• first Archaebacteria

• Lyme disease

• first sulfur metabolizing bacterium

• first bacteria to cause cancer

• resistant to radiation, starvation, ox stress



Genome sequencing

• DOE – Joint Genome Institute

– http://www.jgi.doe.gov/

– Numerous advances in sequencing technology

• Increased pass rate from ~70% to > 90%

• Lowered cost nearly 3 fold


The human genome

• In Feb 12 2001, Celera and Human Genome project published “draft” human genome sequencs

– Celera -> 39114

– Ensembl -> 29691

– Consensus from all sources ~30K

• Number of genes

– C. elegans – 19,000

– Arabidopsis 25,000

• Predictions had been from 50-140k human genes

– What’s up with that?

– Are we only slightly more complicated than a weed?

– How can we possibly get a human with less than 2x the number of genes as C. elegans

– Implications?

• UNRAVELING THE DNA MYTH: The spurious foundation of genetic engineering, Barry Commoner, Harpers Magazine Feb, 2002


The human genome

• The answer – Sloppy science

– Gene sets don’t overlap completely

– Floor is 42K

– 128,826 UniGene clusters from ESTs

= 42113


The human genome

• How finished is the human genome sequence?

– Draft sequence to high coverage

– Chromosome by chromosome finishing now

• Chr 22 – 1999

• Chr 21 – 2000

• Chr 20 – 2001

• Chr 15 – 2003

• Knowing what we know now – how to approach a large new genome?

– Xenopus tropicalis (about ½ human)

– BAC end sequencing

– Whole genome shotgun

– Gaps closed with BACS

– 8 x coverage by end of 2004

– Finishing dependent on additional funding


DNA Sequence analysis

• Complete DNA sequence

– 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)


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?



• 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



• 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



• nested deletion strategies - make sequential deletions from one end of the clone– cut, close and sequence

• 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



• nested deletion strategies (contd)

– cut, close and sequence (contd)

• advantages

– fast

– simple

– efficient

• disadvantages

– limited by restriction site availability in vector and insert

– need to make a restriction map

– BAL31 mediated deletions (archaic)

• digest insert from both ends with BAL31

• repair, subclone and sequence

• advantages

– was once the only way to make progressive deletions

• disadvantages

– bidirectional

– can’t protect -> must reclone

• applications

– no longer used

– superseded by ExoIII-mediated deletion cloning



• nested deletion strategies (contd)

– Exonuclease III-mediated deletion



– Exonuclease III-mediated deletion (contd)

• 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



• 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



• Shotgun sequencing

– NOT invented by Craig Venter

• Messing 1981 first description of shotgun

• Sanger lab developed current methods in 1983

– approach

• blast genome into bite sized chunks

• clone these chunks

• sequence

• assemble the whole mess with a computer

– idealized shotgun strategy

– A priori difficulties

• how to get nice uniform distribution

• how to assemble fragments

• what to do about repeats?

• How to minimize sequence redundancy?









• 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 $$• why does redundancy matter?

– Finished sequence today costs about $0.50/base



– Mapping by fingerprinting

– Mapping by hybridization



• Map as you go



– Whole genome shotgun sequencing (Celera)

• premise is that rapid generation of draft sequence is valuable

• why bother trying to clone and sequence difficult regions?

– Basically just forget regions of repetitive DNA - not cost effective

• using this approach, genome is alleged to be 90% finished

– rule of thumb is that it takes at least as long to finish the last 5% as it took to get the first 95%

• problems

– sequence may never be complete as is C. elegans

– much redundant sequence with many sparse regions and lots of gaps.

– Fragment assembly for regions of highly repetitive DNA is dubious at best

– “Finished” fly and human genomes lack more than a few already characterized genes


Useful software for molecular biology


Useful software for molecular biology (contd)

• NCBI - main information and analysis resource

– indispensable resource









• Why pay Celera?

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BioSci 203 lecture 22 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 22 - cDNA library screening &sequence characterization