Recombinant DNA II

Making, screening and analyzing

cDNA clones
Genomic DNA clones
 cDNA clones are copies of mRNAs
• Much of the genomic DNA is not expressed
as mRNA
• Many issues about gene function are best
addressed by examining the product that
they encode.
• The cDNA copies of mRNA contain primarily
sequences that encode protein.
• Therefore, cDNA clones are useful for many
studies of gene function.
 Construction of cDNA clones
• Use the enzyme reverse transcriptase to
copy mRNA into complementary DNA,
called cDNA. This is equivalent to the
template strand of the duplex DNA.
• Use a DNA polymerase to copy that cDNA
into the nontemplate (message
synonymous) strand.
• Insert the duplex cDNA product into a
cloning vector and propagate in a host, e.g.
E. coli.
 cDNA: first strand synthesis
mRNA 5’ AAAAAAA 3’
Anneal oligo-dT primer TTTTT
AAAAAAA 3’
TTTTT 5’
Reverse transcriptase:
RNA-directed DNA polymerase dNTPs
RNase H
AAAAAAA 3’
TTTTT 5’
Hydrolyze remaining RNA with base

TTTTT 5’
Product is complementary DNA, called cDNA. It is equivalent
to the template strand of the duplex DNA.
 cDNA: second strand synthesis
Problem: How to get a primer for 2nd strand synthesis?
cDNA TTTTT 5’
Terminal deoxynucleotidyl transferase dCTPs

CCCC TTTTT 5’

Ligate an adaptor to the 3’ end 5’ GGGG

3’
5’ GGGG
3’ CCCC TTTTT 5’
DNA polymerase dNTPs
5’ GGGG AAAAA 3’
3’ CCCC TTTTT 5’
Duplex cDNA
 Ligate duplex cDNA into a plasmid
Duplex cDNA
5’ GGGG AAAAA 3’
3’ CCCC TTTTT 5’

Restriction endonuclease Cut the adaptor

GGGG AAAAA
CCCC TTTTT

Ligate duplex cDNA into a plasmid

Transform the population of cDNA plasmids into bacteria.

Result is a cDNA library.
 Limitations of cDNA synthesis
• First strand synthesis often does not go to
completion.
– Individual cDNA clones will frequently have the
reverse complement of only part of the mRNA.
– Multiple cDNA clones from a single mRNA will
be present in the library
• Priming second strand synthesis is inefficient
– Some methods necessarily result in the loss of
sequences at the 5’ end of the nontemplate
strand
 How do you find a cDNA clone from
the desired gene?
• A cDNA library has >100,000 individual
clones.
• It contains copies of as many as 50,000
different mRNAs .
• The frequency of occurrence of a cDNA
from a given gene reflects the abundance of
the mRNA for that gene.
• Try to find correct 1 clone in about 100,000.
 Strategies for screening cDNA clones
• Brute force screen for abundant cDNAs.
• Hybridization with a gene-specific probe.
• Express the cDNA in the host cell (i.e. make
a functional protein product)
– Specific antisera
– Labeled ligand to a receptor
– Assay for a function (complementation)
• Differential analysis
 Screening by hybridization

Each bacterial Filter replica of

colonies contains a DNA in colonies
single type of cDNA Hybridize with a
plasmid labeled DNA from
gene of interest

Detect by
autoradiography
Screening for an expressed product

Filter replica of
protein in colonies

Bind an antibody
specific for the
protein of interest
Detect the bound
antibody with an
enzymatic assay
(generating color
or light).
 Expression screening in eukaryotic cells

“transfect”
introduce cDNA
+
plasmids into
cells Epo
Cell line that
Expression A transformed cell line
needs a
library: that expresses the
cytokine (e.g.
cDNA inserts in Epo receptor will now
IL-3) to grow.
a vector grow in Epo without
Has no Epo
that will drive IL-3. The plasmid with
receptor, will
expression the Epo receptor
not grow in
in eukaryotic cDNA can be isolated
Epo.
cells from this cell line.
 Differential analysis
• Instead of looking for one particular cDNA, look for
cDNAs from all genes whose expression differs in
the process under study
– Differentiation from mesoderm to muscle
– Response to different nutrients
– Progression through S phase of the cell cycle
• Methods:
– Subtractive hybridization
– Differential display
– Hybridization to massively parallel arrays of cDNAs.
 Differential analysis applied to
muscle differentiation

myocytes
(muscle)
adipocytes (fat)
mouse 10T1/2
cells chondrocytes
multipotential 5-azacytidine (cartilage)
 Subtractive hybridization
10T1/2 myocyte

mRNA

mRNA *cDNA (radiolabeled)

anneal with mRNA in excess

mRNA-*cDNA duplexes + *cDNA + mRNA
Should contain cDNAs for Should contain cDNAs
all mRNAs in common specific for cells
between the 2 cell types differentiating into muscle

Repeat mRNA-*cDNA duplexes bind to

a HAP column
column
few
cycles
*cDNA + mRNA elute

Use the labeled *cDNA to Is unlabeled, will

hybridize to a library of not interfere with
cDNA clones from 10T1/2 subsequent
-derived myocytes steps.
 Differential display of RT-PCR products

• Make cDNA from all mRNA in the two different

cellular states (RT = reverse transcriptase).
• Use several sets of PCR primers to amplify a
representative sample of all the cDNAs.
• Resolve those RT- PCR products on a gel.
• Find the products that are present in only one of
the two cellular states being compared.
• Try to isolate the corresponding gene.
Sequence everything, find function later
• Determine the sequence of hundreds of
thousands of cDNA clones from libraries
constructed from many different tissues and
stages of development of organism of
interest.
• Initially, the sequences are partials, and are
referred to as expressed sequence tags
(ESTs).
• Use these cDNAs in high-throughput
screening and testing, e.g. expression
microarrays (next presentation).
 Genomic DNA clones

• Clones of genomic DNA contain fragments of

chromosomal DNA. They are used to:
– obtain detailed structures of genes
– identify regulatory regions
– map and analyze alterations to the genome,
e.g. isolate genes that when mutated cause
a hereditary disease
– direct alterations in the genome
– sequence the genome.
Construction of libraries of genomic DNA

Partially
digest with
restriction
endonuclease
,
select ca.
20,000 bp RE RE
fragments
Total nuclear DNA from an
organism with, e.g., 3 billion bp
in a haploid genome
RE RE

Mix genomic DNA fragments

with a DNA from a cloning
vector (e.g. lambda) and ligate

cos
... ...
cos cos cos

Package the
concatameric DNA
into phage particles
in vitro
 BAC vectors for large DNA inserts
promoter
S E SacB+: SacBII encodes levansucrase,
Cm(R) E
which converts sucrose to levan,
SacBII a compound toxic to the bacteria.
pBACe3.6
oriF 11.5 kb

Cut with restriction enzyme E, remove “stuffer”

Ligate to very large fragments of genomic DNA

promoter Large insert, 300kb

SacBII
S

Cm(R) SacB-: No toxic levan produced on sucrose

oriF
media: positive selection for recombinants.

Not to scale.
 How many clones make a representative library?

• P = probability that a gene is in a library

• f = fraction of the genome in a single recombinant
• f = insert size/genome size
• For N recombinants, 1-P = (1-f)expN
• ln(1-P) = N ln(1-f)
• N = ln(1-P) / ln(1-f)
• For a lambda library with an average insert size of 17 kb
and a genome size of 3 billion bp, then one needs a library
of 800,000 clones to have a probability of 0.99 of having all
genes in the library.
• For a BAC library, with an average insert size of 300 kb
and a genome size of 3 billion bp, then the library size
required for P=0.99 is reduced to about 46,000 clones.
Screening libraries of genomic clones

Genomic library (ca. 800,000 recombinant phage) plus host E. coli

This screen requires 40 plates with 20,000 phage on each plate.

Pour onto
plate and
incubate
Transfer DNA
overnight
from the plaques
onto a membrane
(e.g. nylon)
Each plaque has
DNA from a
different
segment of the
genome * *
* *

*
Expose to X-ray film
to generate an
autoradiogram
 Sequence everything: genomics
• Instead of screening for one gene at a time,
an entire genome can be sequenced, and
one can use experimental and bioinformatic
approaches to find many (all?) genes of
interest.
• Made possible by
– Substantial increases in speed of sequencing
– Larger insert libraries for larger genomes
– Combination of hierarchical sequencing (based
on maps) and whole genome shotgun
sequencing