1. Bio Sci 203 Lecture 19 - cDNA library screening
Bruce Blumberg
office Bio Sci II
lab 5427 (x46873), 5305 (x43116)
office hours Wednesday 1-2.
Link is also main class web site
Today
wrap up cDNA library construction
cDNA library Screening, a.k.a. how do I clone my gene?
BioSci 203 lecture 19 page 1 ©copyright Bruce Blumberg All rights reserved

2. Construction of cDNA libraries
What is a cDNA library?
Collection of DNA copies representing the expressed mRNA population of a cell, tissue, organ or embryo
what are they good for?
Identifying and isolating expressed mRNAs
functional identification of gene products
cataloging expression patterns for a particular tissue
EST sequencing and microarray analysis
BioSci 203 lecture 19 page 2 ©copyright Bruce Blumberg All rights reserved

3. Determinants of library quality
What constitutes a full-length cDNA?
Strictly it is an exact copy of the mRNA
full-length protein coding sequence considered acceptable for most purposes
mRNA
full-length, capped mRNAs are critical to making full-length libraries
cytoplasmic mRNAs are best
1st strand synthesis
complete first strand needs to be synthesized
issues about enzymes
2nd strand synthesis
thought to be less important than 1st strand (probably not)
choice of vector
plasmids are best for EST sequencing
phages are best for manual screening
how will library quality be evaluated
test with 2, 4, 6, 8 kb probes to ensure that these are well represented
BioSci 203 lecture 19 page 3 ©copyright Bruce Blumberg All rights reserved

4. mRNA is isolated from source of interest
cDNA synthesis
Scheme
mRNA is isolated from source of interest
1-2 ug is denatured and annealed to primer containing d(T)n
reverse transcriptase copies mRNA into cDNA
DNA polymerase I and Rnase H convert remaining mRNA into DNA
cDNA is rendered blunt ended
linkers or adapters are added for cloning
cDNA is ligated into a suitable vector
vector is introduced into bacteria
Caveats
there is lots of bad information out there
much is derived from vendors who want to increase sales of their enzymes or kits
all manufacturers do not make equal quality enzymes
most kits are optimized for speed at the expense of quality
small points can make a big difference in the final outcome
BioSci 203 lecture 19 page 4 ©copyright Bruce Blumberg All rights reserved

5. cDNA synthesis (contd)
Preparation of mRNA
want minimum of non poly A+ mRNAs
affinity chromatography oligo d(T) or (U)
Oligo d(T)30 latex (Nippon Roche) works best overall (a.k.a. OligoTex Qiagen)
2 successive runs gives ~90% pure A+ mRNA
denaturation of mRNA
critical step
most protocols use heat denaturation
CH3HgOH is method of choice for best libraries
First strand synthesis
lots of misinformation about enzymes
reverse transcriptase contains 2 subunits
polymerase
RNase H
RNase H subunit is critical for processivity of the enzyme!
Manufacturers want you to buy MMLV RNase H- RT because it is cloned and almost free to make
best enzyme for 1st strand synthesis is AMV RT from Seikagaku America
But not best overall
thought that 1st strand is main failure point in cDNA synthesis - NOT
great improvement in 1st strand synthesis is addition of 0.6M trehalose to reaction
allows rxns to run at ~60° C
BioSci 203 lecture 19 page 5 ©copyright Bruce Blumberg All rights reserved

6. cDNA synthesis (contd)
2nd strand
must remove mRNA
best way is with RNAse H so that fragments serve as primers for DNA pol I
Gubler and Hoffman (1983) Gene 25, 263
in my experience, 2nd strand synthesis is the point of failure in cDNA
virtually all kits shortcut this step (1-2 hrs)
should be overnight
recent improvement is to use thermostabile RNAse H, DNA ligase and DNA polymerase to maximize production of 2nd strand.
BioSci 203 lecture 19 page 6 ©copyright Bruce Blumberg All rights reserved

7. cDNA synthesis (contd)
BioSci 203 lecture 19 page 7 ©copyright Bruce Blumberg All rights reserved

8. cDNA synthesis (contd)
Cloning
after 2nd strand is made, the ends must be blunted and linkers or adapters added
usually T4 DNA polymerase
perfect cDNAs will retain 2-20 bp of RNA at the 5’ end.
Linkers can not be added to this by any DNA ligase!
But T4 RNA ligase can ligate DNA-RNA and stimulates blunt end ligation 10x
no commercial products use T4 RNA ligase so it is no wonder that full-length cDNAs are lost
if internal restriction sites have not been protected, they need to be methylated now before linkers are added.
Most methylase preps are not clean
BioSci 203 lecture 19 page 8 ©copyright Bruce Blumberg All rights reserved

9. Full-length mRNA isolation and cDNA synthesis
Ways to capture cap structures and presumably full-length mRNAs
affinity chromatography with eIF-4E (cap binding protein a.k.a. Capture
selection with antibody to cap structure
oligo capping
biotinylated cap trapper
5’ oligo capping - Maruyama, K., and Sugano, S. (1994). Gene 138,
Principle is that uncapped mRNAs are dephosphorylated so that they cannot be ligated
cap structure is removed leaving only previously capped mRNAs with 5’ PO4
RNA ligase can ligate a 5’-OH oligo to the 5’ end of the mRNA
This can be used to prime 2nd strand synthesis
BioSci 203 lecture 19 page 9 ©copyright Bruce Blumberg All rights reserved

10. Full-length mRNA isolation and cDNA synthesis (contd)
5’ oligo capping (contd)
advantages
very simple
no homopolymeric regions to worry about
can put arbitrary sequence at 5’ end.
Enables custom vector construction
also enables PCR to make driver for normalization
disadvantages
cap trapper paper claims this method only gives 70% full-length cDNAs
high quality TAP is not easy to find
original paper used PCR between 5’ and 3’ primer to make cDNAs
PCR => bias!
BioSci 203 lecture 19 page 10 ©copyright Bruce Blumberg All rights reserved

11. Full-length mRNA isolation and cDNA synthesis (contd)
Cap trapping Carninci, P. et al. (1996) Genomics 37:
Principle is that a biotin residue is chemically added to the cap structure
approach
1st strand cDNA is synthesized
treatment with RNAse I cuts any cDNA:mRNA duplexes which are not absolutely complete
complete cDNAs are isolated by streptavidin chromatography
RNA is hydrolyzed
cDNA is tailed with dG
2nd strand synthesis is primed with dC
adapter added
cloned
advantages
claimed to give 90% recovery of full-length cDNAs
lots of history at RIKEN
disadvantages
homopolymeric region may make functional analysis of cDNAs difficult or impossible
many steps -> points of failure
BioSci 203 lecture 19 page 11 ©copyright Bruce Blumberg All rights reserved

12. Full-length mRNA isolation and cDNA synthesis (contd)
BioSci 203 lecture 19 page 12 ©copyright Bruce Blumberg All rights reserved

13. Full-length mRNA isolation and cDNA synthesis (contd)
Cloning of cDNAs
most methods require linker or adapter addition followed by restriction digestion
relies on methylation to protect internal sites or use of rare cutters
A new alternative is ExoIII-mediated subcloning
no methylation
no restriction digestion
no ligation
no multimerization of vector or inserts
100% oriented
BioSci 203 lecture 19 page 13 ©copyright Bruce Blumberg All rights reserved

14. Vectors for cDNA cloning
Plasmids vs phage
phage are preferred for high density manual screening with all types of probes
plasmids are preferred for functional screening
microinjection
transfection
panning
phage packaging and infection is more routinely efficient than electroporation
10-100x better than best transformation frequency
what will the library be used for ?
Consider the intended use as well as other contemplated uses
will the library go to an EST project?
Plasmid
will it be screened manually
phage
or arrayed and screened on high density filters
plasmid
will we normalize it?
Probably plasmid
BioSci 203 lecture 19 page 14 ©copyright Bruce Blumberg All rights reserved

15. Vectors for cDNA cloning (contd)
Analysis of cDNAs obtained
rate limiting step in clone analysis is getting them into a usable form
usually a plasmid
cloning is tedious, particularly if one has many positives
some tricks can be used but this is still the bottleneck
in about 1985 or so, Stratagene introduced lambda ZAP
phage with an embedded plasmid and M13 packaging signals
plasmid can be automatically excised by adding a helper phage
gene II protein replicates plasmid into ss phagemid which is secreted
this was a major advance and many phage libraries today are made in ZAP or its derivatives
early protocols had problems with helper phage but this has been overcome
later, others developed a Cre-lox based system
instead of M13 used loxP sites.
When Cre recombinase is added, recombination between the loxP sites excises a plasmid
both methods work very well and make analysis of many clones very straightforward
BioSci 203 lecture 19 page 15 ©copyright Bruce Blumberg All rights reserved

16. Vectors for cDNA cloning (contd)
BioSci 203 lecture 19 page 16 ©copyright Bruce Blumberg All rights reserved

17. Vectors for cDNA cloning (contd)
BioSci 203 lecture 19 page 17 ©copyright Bruce Blumberg All rights reserved

18. mRNA frequency and cloning
mRNA frequency classes
classic references
Bishop et al., 1974 Nature 250,
Davidson and Britten, 1979 Science 204,
abundant
10-15 mRNAs that together represent 10-20% of the total RNA mass
> 0.2%
intermediate
1,000-2,000 mRNAs together comprising 40-45% of the total
% abundance
rare
15,000-20,000 mRNAs comprising % of the total
abundance of each is less than 0.05% of the total
some of these might only occur at a few copies per cell
How does one go about identifying genes that might only occur at a few copies per cell?
BioSci 203 lecture 19 page 18 ©copyright Bruce Blumberg All rights reserved

19. Normalization and subtraction
How does one go about identifying genes that might only occur at a few copies per cell?
Improve your chances by altering the representation of the cDNAs in a library or population
Normalization - process of reducing the frequency of abundant and increasing the frequency of rare mRNAs
Bonaldo et al., 1996 Genome Research 6,
normalization is claimed to bring all cDNAs into the same order of magnitude abundance, i.e., within 10 fold of each other
rarely works this well.
More commonly, abundant genes are reduced 10 fold and rare ones increased 3-10 fold.
Intermediate class genes do not change much at all
Approach
make a population of cDNAs single stranded
tester
hybridize with a large excess of cDNA or mRNA to Cot =5.5
driver
Cot value is critical for success of normalization
5-10 is optimal
higher values are not better
BioSci 203 lecture 19 page 19 ©copyright Bruce Blumberg All rights reserved

20. Normalization and subtraction (contd)
Approach (contd)
various approaches to make driver
use mRNA - may not be easy to get
make ssRNA by transcribing library
make ssDNA by ExoIII treating inserts digested from plasmid library
PCR amplification of library
experience has demonstrated that the best approach is to use driver derived from the same library by PCR
rapid, simple and effective
other approaches each have various technical difficulties
see the Bonaldo review for details.
What are normalized libraries good for?
EST sequencing
gene identification
biggest use is to reduce the number of cDNAs that must be screened
good general purpose target to screen
subtracted libraries are useful but limited in utility
BioSci 203 lecture 19 page 20 ©copyright Bruce Blumberg All rights reserved

21. Normalization and subtraction (contd)
Subtraction - the process of removing cDNAs (mRNAs) expressed in two populations leaving only those that are differentially expressed
review Sagerström et al. (1997) Ann Rev. Biochem 66,
Plus minus screening St. John and Davis (1979) Cell 16,
Hybridize the same library with probes prepared from two different sources and compare the results
standard screening selects for presence of a spot
may underestimate rare mRNAs so reverse method is sometimes used
I.e., select for absence of a signal
example - hybridize a liver cDNA library with probes prepared from normal and cancerous liver
standard +/- -> select for genes present in cancer or normal
reverse +/- -> select for genes absent
typically requires >10 fold difference in expression levels using standard methods -> not widely used
microarray analysis is a refined version of +/- screening
fluorescence ratios give good internal standards
more precise quantitation
increased sensitivity
BioSci 203 lecture 19 page 21 ©copyright Bruce Blumberg All rights reserved

22. Normalization and subtraction (contd)
Subtractive screening - Sargent and Dawid (1983) Science 222,
Make 1st strand cDNA from a tissue and then hybridize it to excess mRNA from another
larger Cot is best
remove double stranded materials -> common seqs
make a probe or library from the remaining single stranded cDNA
fold more sensitive than +/- screening
BioSci 203 lecture 19 page 22 ©copyright Bruce Blumberg All rights reserved

23. Normalization and subtraction (contd)
Subtractive screening (contd)
benefits
sensitive
can simultaneously identify all cDNAs that are differentially present in a population
good choice for identifying unknown, tissue specific genes
drawbacks
easy to have abundant housekeeping genes slip through
multistage subtraction is best
in effect normalize first, then subtract
libraries have limited applications
may not be useful for multiple purposes
rule of thumb
make a high quality representative library from a tissue of interest
save subtraction and other fancy manipulations for making probes to screen such libraries with
unlimited screening
easy to use libraries for different purposes, e.g. the liver library
hepatocarcinoma
cirrhosis
regeneration specific genes
BioSci 203 lecture 19 page 23 ©copyright Bruce Blumberg All rights reserved