Transcript 4 - cellbiochem.ca

Recombinant DNA Technology
CHMI 4226 E
Week # 4
January 26, 2009
cDNA libraries
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Genomes, Genes and Organisms
• The genome contains
all the genetic
information necessary
for the formation of a
complete, functional
living organism;
• Generally, the more
evolved an organism,
the bigger the genome,
and the greater the
number of genes.
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Genomes, Genes and Organisms
• However, a few
questions arise
upon closer
examination:
– D. melanogaster
(the fruit fly), C.
elegans (nematode),
A. thaliana (mustard
plant) and humans
have similar number
of genes in their
genomes;
– The same set of
genes is sufficient
for the generation of
a wide diversity of
organs and tissues
(e.g. brain vs liver vs
skin).
Species
Human
Fruit fly (Drosophila
melanogaster)
Baker's yeast
(Saccharomyces cerevisiae)
Worm (Caenorhabditis
elegans)
E. coli
Arabidopsis (Arabidopsis
thaliana)
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Size of
genome
Number
of genes
2.9 billion
base pairs
20,0025,000
120 million
base pairs
13,601
12 million
base pairs
6, 275
97 million
base pairs
19,000
4.1 million
base pairs
4,800
125 million
base pairs
25,000
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Genomes, Genes and Organisms
•
These observations can be
explained by the following:
– Differences in gene
expression (both qualitative
and quantitative);
– A substantial fraction of
genes in higher vertebrates
can code for multiple mRNA
species through alternative
splicing, giving rise to a great
variety of protein products.
– Alternative splicing is
differentially regulated in
different cell types.
http://www.exonhit.com/alternativesplicing/pages/diagrams/figure2.htm
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Genomes, Genes and Organisms
• Thus, in order to understand the molecular basis
of function (proliferation, differentiation, death),
one must know which genes are expressed in
which cell/tissue type.
• This is often done through the use of cDNA
libraries.
• cDNA (complementary DNA): a DNA copy of an
mRNA (thus, contains the coding info of the
genome – so, NO introns).
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Genomes, Genes and Organisms
• cDNA libraries:
– A mixture of cDNAs each cloned in a vector (usually
a phage-based vector);
– Each cDNA library is prepared from RNA isolated
from a specific tissue/organ, and therefore
represents the mRNA population (qualitatively and
quantitatively) of that cell/tissue at the time of RNA
extraction;
– Because cDNA libraries contain thousands of different
cDNAs, strategies must be designed to isolate the
cDNA of interest (library screening).
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cDNA libraries – basic steps
Cell/Tissue
Library screening
RNA isolation
Characterize clones
Reverse
transcription
cDNA synthesis
Clone cDNA in vector
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RNA isolation
Cell/Tissue
Lysis in acid Guanidium thiocyanate Buffer
(chaotropic agent – very good at denaturing RNAses)
Acid Phenol extraction
-Solubilizes proteins and DNA
-RNA stays in GT buffer
Ethanol precipitation
RNA analysis on agarose/
formaldehyde gel
Total RNA is: - 85% ribosomal RNA
- 15 % transfer RNA
- 1-3% mRNA
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Poly (A+) RNA isolation
mRNA’s 3’ end is extended after transcription by a tail of polyadenosine (poly A tail).
No other type of RNA has that kind of modification.
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Poly (A+) RNA isolation
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cDNA synthesis
Radioactive dCTP is
added as tracer.
RNAse H cleaves only the RNA
in an RNA/DNA duplex.
RNA fragments left by RNAse H
act as primers for the synthesis of
the coding strand of the cDNA.
EcoR I methylase add CH3 groups to
GAATTC sites and prevent EcoR I
from cutting the cDNA.
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cDNA synthesis
Size fractionation is necessary in order to ensure that most of the
cDNAs in the library are large enough to contain a complete
mRNA.
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Phage lambda
Lytic cycle
Lysogenic cycle
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Phage lambda
Clear lysis plaques of phage lambda grown on a lawn of
E. coli bacteria
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Phage lambda
Genes for phage
insertion and
excision
lgt11
lgt10
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cDNA libraries
WITH helper phage = pBluescript
NO helper phage = lytic cycle
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cDNA libraries
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Screening
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Screening
• Radioactive DNA/RNA probes:
–
–
–
–
Partial (incomplete) cDNA;
cDNA from a similar molecule from same species;
cDNA from another species;
Oligonucleotide (deduced from amino acid
sequence);
• Antibodies;
• Functional screening (according to the
properties of the protein).
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cDNA labeling
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DNA hybridization
• Several factors influence
the rate of DNA
hybridization
95oC
– Nucleotide sequence
• Relative abondance of AT
vs GC
• Length
• Presence of repetitive
sequences
• Extent of sequence
complementarity
(mismatches)
–
–
–
–
Temperature
Salt concentration
pH
Additives (e.g. formamide)
Hybridization
(e.g. Tm-5oC)
Wash-out unhybridized probe
Detection by autoradiography or phosphorimaging
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Detection of a radioactive signal
Autoradiography
•
The X-ray film is a photographic
emulsions consisting of silver
halide crystals in suspension in
a clear gelatinous phase.
•
β-particle or a γ-ray emitted by
a radionuclide convert Ag+ ions
to Ag atoms, which leave a dark
precipitate.
•
Variables affecting the darkness
of the spots include:
– Radioactivity of the sample
– Time of exposure
– Temperature:
• E.g.: exposing film with 32Plabeled sample at -80oC instead
of room temperature increases
the signal several fold.
Signal
saturates
rapidly
Increasing amounts of
radioactivity in sample
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Detection of a radioactive signal
Autoradiography
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Detection of a radioactive signal
Storage Phosphor systems
32P/35S
 b emitters
BaFBr:Eu2+ + b-  BaFBr:Eu3+
BaFBr:Eu3+
↓hn (633nm)
BaFBr:Eu2+
+
hn (390 nm)
http://radiographics.rsnajnls.org/content/vol27/issue3/images/large/g07ma02g03x.jpeg
Ref: R. Switzer and L. Garrity. Experimental Biochemistry – Theory and exercises in fundamental methods, 1999, 3rd ed. W.H . Freeman and Co.
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Detection of a radioactive signal
Storage Phosphor systems
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http://las.perkinelmer.com/ProductCatalogTrack/Templates/EnlargeImgPopUp.aspx?ImagePath=%2fContent%2fImages%2flargeImages%2fC431220.jpg&AltText=c431220
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Detection of a radioactive signal
Storage Phosphor vs autoradiography
• Advantages of SP
vs Autorad:
– Dynamic range:
• >105 fold for PS
• 10 fold for autorad
– Exposure times 10
to 250 shorter
– Screens are reusable indefinitely
Pg A.3A.8
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Detection by autoradiography
Clones!!!
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Other probes:
Expressed Sequence Tags
mRNA isolation
Upload data in
EST database
Reverse
Transcriptase
Sequence millions
of cDNA clones
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cDNA mixture
Clone cDNAs into
plasmids
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Other probes:
Expressed Sequence Tags
• Anonymous cDNA sequences
• Obtained from the random, large scale sequencing of cDNA clones
• Very useful:
– Qualitative and quantitative representation of the mRNAs expressed in
a particular cell line (the transcriptome);
– Obtain data on the transcriptome of a specific organism under specific
conditions (develpment, tissue-specific expression, stress-related,
pathology-related);
– Data on alternative forms of an mRNA (results of alternative splicing)
– Makes Blasts searches easier: a hit on an EST will give you the
nucleotide sequence of your cDNA!!
• However: Mostly limited to model organisms (human, rat, mouse,
fruit fly, nematode, A. thaliana, S. cerevisiae, S. pombe, a few
bacterial species).
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Other probes: Riboprobes
For riboprobe synthesis, an RNA version of the DNA probe is made by
in-vitro transcription in the presence of NTPs, an RNA polymerase (T7 or
T3 in this case), the DNA fragment of interest, and a radioactive nucleotide
(e.g. a32P-ATP).
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Other probes: Oligonucleotides
• THESE ARE NOT
PRIMERS!!!!!!!!!!!!!!!!!!!
• The oligonucleotides
can then be labeled with
T4 DNA kinase in the
presence of g32P-ATP
• When designing an
oligonucleotide from an
amino acid sequence,
remember:
– The genetic code is
degenerate;
– Some codons are more
frequently used than
others;
– Keep the degeneracy
to a minimum.
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Codon usage in human
http://www.kazusa.or.jp/codon/
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Screeening
with
antibodies
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Expression
Screening
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Screening
via cell
panning
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cDNA clones!
AmpR
Ori C
Restriction mapping
Complete?
Sequencing
Sub-cloning
probe
5’/3’ RACE
Mutagenesis
mRNA expression
Identification
Expressed sequence tag
(EST)
Blast
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5’ and 3’ Rapid Amplification of
cDNA Ends (RACE)
5’ RACE
3’ RACE
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