Download Molecular Methods - Roswell Park Cancer Institute

Basic Molecular Methods
“A historical look genetic analysis”
Rosalind Franklin
(Image: wikipedia.com)
Adam Kisailus, Ph.D.
Molecular Methods
What will be presented: experimental
methods used for studying the molecular
biology of cancer
Goal of this class: Obtain an appreciation
of practical application of molecular
techniques
Molecular analysis of BRCA
 Hereditary breast cancer versus sporadic breast
cancer
 Germ line mutations to BRCA gene correlated
with breast cancer susceptibility
 Gene was partially mapped in 1992
 Gene was “discovered” in 1996
 Characterized as a tumor suppressor gene
which plays a role in cell cycle inhibition (1997Present)
 2002- Epigenetically regulated
Anatomy of a gene
Gene is a coding unit for a protein
Exon1
Intron
Exon 2
Exon: encodes protein
Intron: does not encode protein
The central dogma
Exon1
replication
Exon 2
transcription
Exon1
DNA (gene)
reverse- transcription
Exon 2
mRNA
translation
protein
The central dogma in cancer cells
Mutation
Exon1
replication
Exon 2
DNA (oncogene)
transcription
Exon1
aberrant mRNA
translation
Tumor suppressor
-orTumor promoter
Identification of the BRCA mutation
Mutation of BRCA correlates with disease
onset JM Hall et al Am. J Hum. Genet. 1992 June; 50(6):1235-1242
BRCA is defined as a tumor suppressor
Casey et al Hum Mol Genet. 1993 Nov;2(11):1921-7
The central dogma in cancer cells
Mutation
Exon1
replication
Exon 2
DNA (oncogene)
transcription
Exon1
aberrant mRNA
Translation
Tumor suppressor
-orTumor promoter
1992-1996: Mapping the BRCA gene
and mutation identification
RFLP analysis
Southern blotting
PCR
Kary Mullis- (Google images)
Properties of DNA
 Complementary base
pairing
 Anneals/denatures
under varying
temperature conditions,
 Negatively charged
phosphate backbone
 Semi-conservative
replication
Source: Google Images
Molecular biologists aren’t inventors,
they’re opportunists
How can we harness these properties of
DNA to study our gene?
Restriction Fragment Length
Polymorphisms (RFLP) analysis
Single nucleotide polymorphisms (SNPs)
Used as genetic “fingerprint”
Polymorphisms can be mutations in
oncogenes
Detection of polymorphisms by 2
component method: (1) restriction
enzyme digest and (2) Southern blot
analysis
Restriction Enzymes
 Restriction Digest Enzymes
Bacterial origin, defense mechanism for bacteria to
fragment invading phage (bacterial virus)
Recognize and cleave specific palindromic site on DNA
Very straight forward usage, practically no problems
except for STAR activity.
Resulting digested DNA is a restriction fragment
Eco RI:
5’-ATGCGC G-A-A-T-T-C GGCCT-3’
3’-TACGCG C-T-T-A-A-G CCGGA-5’
Restriction fragments and sticky ends
Eco RI:
5’-ATGCGC G-A-A-T-T-C GGCCT-3’
3’-TACGCG C-T-T-A-A-G CCGGA-5’
Eco RI:
5’-ATGCGC G-3’
3’-TACGCG C-T-T-A-A-5’
Can pair with
Corresponding DNA
bases
Single Nucleotide Polymorphism (SNP)
Restriction enzyme no longer recognizes cut sequence.
And does not cut.
Eco RI:
5’-ATGCGC G-A-G-T-T-C GGCCT-3’
3’-TACGCG C-T-C-A-A-G CCGGA-5’
RFLP Analysis: Genetic diagnostic
RFLP Analysis of Offspring’s DNA:
1. Cut from human
chromosome with
reference R.E. sites
2. Digested with
mut
BRCA
(Paternal)
BRCA
(Maternal)
specific R.E.s
Restriction digest
mut
2. Probe with
Radio labeled
ssDNA probe
BRCA
(Paternal)
BRCA
(Maternal)
Gel electrophoresis
Gel electrophoresis
Source: Google images
Southern blotting
 Method for analyzing structure of DNA and
identifying mutations within regions of small
sequence repeats
Obtain genomic DNA samples from patient and both
parents.
Digest DNA with restriction enzymes to map mutations
Probe for DNA fragments to visualize genetic profile
Southern Blot analysis of BRCA gene
RFLP Analysis of Offspring’s DNA:
BRCA
mut
BRCA
Paternal (P)
Maternal (M)
Cut sites
Restriction digest
BRCA
mut
BRCA
“DNA probing” (Source:Google Images)
M
P
M
O
P
O
Paternal (P)
Maternal (M)
Denaturation to
ssDNA
and blotting
to membrane
Gel electrophoresis
autoradiography
1997: BRCA gene product analysis
 Study of BRCA 1992-1996
Know it correlates with cancer
Loss of function results in tumorigenesis
 What is its molecular function?
In vitro studies
Clone the BRCA gene for analysis of protein
product
“Characterization of cancer-linked BRCA1-BRCT missense variants and their
interaction with phosphoprotein targets.”
Drikos I, Nounesis G, Vorgias CE.
Department of Biochemistry and Molecular Biology, Faculty of Biology, National
and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15701
Athens, Hellas.
The central dogma
Exon1
replication
Exon 2
transcription
Exon1
DNA (gene)
reverse- transcription
Exon 2
mRNA
translation
protein
Molecular cloning and the birth of
biotech
 1980- Genentech
 Herb Boyer and
Robert Swanson:
“Science meets
business- in a bar.”
 Recombinant insulin1978
 Human Growth
hormone -1985
 1988- Canseco and
Mcgwire the bash bro.
Source: Frederic Larson
Steps of molecular cloning
Prepare cDNA (using RT-PCR)
Prepare plasmid DNA
Insert cDNA into plasmid DNA
Transform bacteria and select for plasmidcontaining bacteria
Confirm cDNA insertion
Polymerase Chain Reaction (P.C.R.)
techniques
PCR: gene product amplification starting
from template DNA
 generate DNA for cloning
RT-PCR: gene product amplification
starting from template RNA (recall the
central dogma)
Generate cDNA for cloning
 measure gene expression quantitative RT-PCR
Screen mRNA for mutations
Polymerase Chain Reaction (PCR)
 Supposedly developed by eccentric scientist
Carey Mullis while he was driving the west coast
highway “tripping on acid”
 Components
DNA polymerase originating from Thermos Aquaticus
(Taq polymerase) to withstand high temperatures
 Polymerases vary by fidelity and range depending on
usage
deoxynucleotides (dCTP, dATP, dGTP, dTTP) in equal
amounts!
Oligo primers: designed to specified gene, length and
GC content important for annealing
Co-enzymes: MgCl and buffer
Properties of DNA
 Complementary base
pairing
 Anneals/denatures
under varying
temperature conditions,
depends on sequence
 Negatively charged
phosphate backbone
 Semi-conservative
replication
P.C.R. Thermocycling
27-33 cycles
+ 5’
3’
- 3’
5’
5’
3’
3’
5’
Taq
5’
3’
Taq
3’
5’
94o “DNA template
strand separation”
53o “Primer annealing”
72o “Primer extension”
RT-P.C.R.: Making cDNA for Cloning
Trizol® extraction of mRNA from normal tissue: mRNA is solubilized
in aqueous phase while organic phases precipitate lipids/protein/DNA
1.) First strand synthesis:
Primer: Oligo (dT)
+reverse transcriptase
mRNA 5’
DNA 3’
2.) Digestion of RNA
By RNaseH
mRNA 5’
AAAA-OH 3’
TTTT 5’
mRNA 5’
AAAA-OH 3’
TTTT 5’
3.) Use mRNA fragments
As primers for DNA pol I
(klenow fragment) for
second strand
synthesis
3.) Second strand synthesis
Completion Pol I.
“5’
“3’
3’ ”
AAAA-OH 3’
TTTT 5’
5’ ”
AAAA-OH 3’
TTTT 5’
cDNA 5’
3’
(Product DNA)
Visualizing P.C.R. Product
 Gel electrophoresis
 Ethidium Bromide
(Source: Google images)
Plasmid DNA
 Plasmid DNA is used for
cDNA expression in
prokaryotic/eukaryotic
cells
 Anti-biotic resistance
selection marker
(1)
 Origin of replication (2)
 Multiple cloning site (3)
 Protein fusion tag (4)
(4)
(3)
(2)
(1)
“Plasmid DNA Map”
(Source: NEB catalogue)
Putting it all together: Cloning of BRCA cDNA
schematic
A.
BRCA cDNA
B
ATG
PCR using
Oligos incorporating
restriction enzyme sites
R
ATG
MCS
E
X
Linearize vector by
E digestion
X
D.
C.
Transformation
of bacteria
R
X
ligation
Selection on
antibiotic
E
Propagation in culture
(cloning)
Confirm BRCA gene is inserted in vector
 PCR check
 Digestion diagnostic
 Sequencing- “Old
School” Method
DeoxyHH
(-OH available Di-Deoxyto make phosphate
Back bone)
GGGGTTTAAG
4 Reactions- contains 4 deoxy-nucleotides
And 1 dideoxy-nucleotide of either A,C,T,G
dATP
dTTP
dGTP
dCTP
ddXTP-PCR stopper, because of dideoxy nature
There is no available –OH group for adding
another base. P32 labeled and will appear as band
on autoradiograph
Polymer chain
+ ddG ddA ddT ddC
PCR products
Run on gel
-
Confirm BRCA gene is inserted in vector
“New school” method of PCR
Take primer and plasmid to core facility
Automatic fluorescent sequencing: Incorporation of dye-labeled
ddNTPs into DNA extension products. Each dye emits a different
wavelength when excited by argon ion laser so that all four
bases are detectable in a single lane of the gel.
GGGGTTTAAG
PCR products
Run on gel
ddATP
ddTTP
ddGTP
ddCTP
Things to consider when cloning a gene
 Restriction enzyme usage
Use an internet software tool like NEB®Cutter to ensure
the restriction sites you select occur nowhere else within
the gene
Use two different restriction enzymes for directionality of
insertion
Always, always, when available use enzymes which
leave sticky ends, avoid blunt-end ligation
http://tools.neb.com/NEBcutter2/index.php
Things to consider when cloning a gene
(cont.)
 Oligo design
 Include a GAG sequence to ensure that the restriction enzyme has
enough DNA “scaffold”
 Make sure that no more than 3 bases of forward and reverse oligos are
complimentary to avoid “primer dimers” http://
www.basic.northwestern.edu/biotools/oligocalc.html
 Temperature of melting (50-55 degrees C) depends on GC content and
length of oligos
Forward primer, use – strand as template: 5’-GAGGAATTCATGCCGTACGGCCGG-3’
Reverse primer, use + strand as template 5’-GAGCTCGAGCTACTTCGGGGCCCAG-3’
15 bases into coding region
+ 5’
- 3’
3’
http://www.basic.northwestern.edu/biotools/oligocalc.html
http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=27886591
Things to consider when cloning a gene
(cont.)
 Usage of shuttle vector
Taq polymerase leaves behind a 5’ T and 3’ A overhang
TOPO shuttle vector has these overhangs and therefore
your PCR product with restriction sites intact can be
easily inserted into the TOPO shuttle vector, propagated
and then cut with restriction enzymes
Cutting PCR product with restriction enzymes not very
efficient
 Ligation conditions
Linearized vector must be gel purified
Use 5 to 10 fold excess amount of insert to vector to
drive ligation reaction
Bacterial Transformation
 Transformation: Inserting plasmid DNA into
bacteria
 DH5-alpha: Suitable cell line for cloning
 Steps involved in transformation:
1.) Inoculate competent cells with plasmid DNA
2.) Heat shock
3.) Recover in LB media without antibiotic
4.) Select for bacteria which uptook plasmid DNA on
media containing antibiotic for selection
Confirming BRCA gene is inserted in
vector
 Extract DNA from
bacteria
 Buffer steps:
 1.) Resuspend overnight
culture of bacteria in
resuspension buffer and
RNase
 2.) Lyse bacteria in SDSa detergent and NaOH a
base
 3.) Neutralize in acidic
buffer of sodium acetate
 4.) Precipitate DNA in 2propanol
 5.) Wash in 70% ethanol
 6.)Resuspend pellet in
TE buffer
1.)
2.)
3.)
http://learn.genetics.utah.edu/units/biotech/
Measuring gene expression levels
RT-PCR
mRNA
Northern blot analysis
mRNA
Western blot analysis
protein
Genes Chromosomes Cancer. 1997 Sep;20(1):53-9.
“Analysis of the BRCA1 and BRCA2 genes in sporadic meningiomas.”
Kirsch M, Zhu JJ, Black PM.
Utilize BRCA cDNA to analyze BRCA gene
expression and structure
Southern blot – gene mutation
replication
and structure analysis
“DNA probe for genomic DNA”
Northern blot- gene expression
“DNA probe for RNA”
Western blot- protein expression
”antibody probe for
protein detection”
Far Western blot- protein interaction
”protein overlay for
protein:protein interaction”
Exon1
Exon 2
DNA (gene)
transcription
Exon1 Exon 2
mRNA
translation
protein
Quantitating BRCA gene expression by
real time-PCR (not RT-PCR)
Quantitation of low amounts of mRNA or low copy number DNA using PCR
relies on the concept that DNA binding dyes (Envision®,Sybr green) fluoresce
when incorporated into double stranded DNA)
Eliminates the need for post-PCR processing or gels.
+ 5’
3’
94o “DNA template strand separation”
- 3’
5’
5’
3’
3’
5’
5’
27-33 cycles
Taq
3’
53o “Primer annealing”
72o “Primer extension”
Digital measurement of
signal.
Northern Blot analysis of BRCA gene
expression in cancer cell line
N
1o
M
N
Transfer mRNA
membrane
Gel electrophoresis
1o
M
N
1o
M
Expose to
film
Probe with P32-labeled
BRCA cDNA
Note: in silico northerns for most genes are available at NCBI.
Auto-radiograph
Protein Analysis: Exploiting properties of
proteins
 Proteins are composed of
-/+ charged, inert, acidic
and basic amino acids
 Amino acid sequences
are antigenic
 Proteins are heat and
organic chemicaldenatured
(Source: Google images)
Protein Electrophoresis
1.
- - -
2.
- -
1 2
SDS binds to amino
acid residues and gives
uniform negative charge
to protein; heat/mercaptoethanol
denatures/linearizes protein
Protein
3.
1 2
-
-
Add protein sample to
SDS-PAGE Gel lane 2.
Electric current
Protein separated
by “APPARENT”
molecular weight
Adapted from molecularstation.com
Western Blot Analysis: Immunodetection
 Immunodetection of protein in cell lysate.
 Components
 Primary antibody: has affinity for target protein
 Monoclonal: Recognizes specific epitope on protein, made in
mouse
 Polyclonal: Antibody made to whole protein typically in rabbit
 Secondary antibody: has affinity for primary antibody
 Typically generated in goat
 Cost-effective
 Signal
 Secondary antibody is conjugated to a signal producing enzyme
or fluor
• Alexa Fluor: Emits light when excited by infrared beam (LiCor)
• Horse Radish Peroxidase: Chemical substrates added which are
metabolized by HRP generating chemiluminescent signal.
Western Blot Analysis:Blotting
2o antibody
Cell lysates
N Ca
-
1o antibody
+
Block
non-specific
Sites on membrane
(Antibody
detection)
N
Ca
Denaturation of protein
And uniform neg. charge. Transfer to membrane
SDS PAGE gel separation by charge.
Signal detection on film
Method for studying gene function controversy:
Knocking out a gene versus overexpressing a
gene
 Overexpression of some genes such as those
which encode scaffolding molecules may result
in artifact; however, in other cases it can provide
function of a protein’s activity
Transient protein expression
Stable protein expression
 Silencing or knocking down gene expression
gives clearer indication of gene function
RNA interference (RNAi)
Transfection of mammalian cells
 Vector
Mammalian expression vectors
Bacterial expression vectors
 Delivery Method
DEAE-dextran- old school method; precipitation
between DEAE (+charge) and DNA, uptake by
pinocytosis-?
Lipofection- use of liposome/DNA complex (Fugene®;
Lipofectamine®)
Electroporation- use of electric current, efficient means,
but kills almost 90% of the cells.
Comparison of mammalian expression and
bacterial expression vectors
MCS
TAG
MCS
Antibiotic
selection
Stable
Transf.
Bacterial expression vector:
Mammalian expression vector
(Source: NEB Catalogue)
2001: Understanding the function of BRCA by expression
“knockdown”
Nature Paper 1998- Andrew Fire and
Craig Mello,
2006 Nobe Laureates
Antisense inhibition of BRCA1 expression and molecular analysis of hereditary
tumors indicate that functional inactivation of the p53 DNA damage
response pathway is required for BRCA-associated tumorigenesis.
Reedy MB, Hang T, Gallion H, Arnold S, Smith SA.
RNAi and siRNA:Andrew Fire and
Craig Mello 1998
 siRNA is
complementary
to mRNA,
forming a double
stranded
complex
resulting in its
degradation and
inhibiting
translation into
protein
Exon1
Exon 2
FAK Gene (DNA)
mRNA
siRNA
NH
protein
COOH
?
signaling
“Central dogma”
degraded
siRNA knockdown of BRCA
 Introduce siRNA for BRCA1 into cancer cell
 Knock down BRCA1 levels, treat with
chemotherapy to determine drug resistance
affects
Oncogene. 2009 Jan 29;28(4):575-86. Epub 2008 Nov 10
“Decreased BRCA1 confers tamoxifen resistance in breast cancer cells by altering
estrogen receptor-coregulator interactions.”
Wen J, Li R, Lu Y, Shupnik MA.
2002-2009: BRCA characterization
and function
Domain mapping
Protein:Protein interactions
Enzymatic activity
“Characterization of cancer-linked BRCA1-BRCT
missense variants and their interaction with
phosphoprotein targets.”
Drikos I, Nounesis G, Vorgias CE.
Department of Biochemistry and Molecular
Biology, Faculty of Biology, National and
Kapodistrian University of Athens,
Panepistimiopolis-Zographou, 15701 Athens,
Hellas.
Characterization of signaling domains by site-directedmutagenesis
Following wholesale knockout of gene
expression we’d like to carefully map a
binding site or activity
Site-directed-mutagenesis enables precise
alterations to amino acids by relying on
design of a pair of oligo primers with
mismatched base pairs.
Site-directed-mutagenesis
CH3
Mammalian
Expression
vector
CH3
MOVE
Not I digestion
Leu Gly Ala Asn Leu
TTA GGA GCT AAT TTA
AAT CCT CGA GCA AAT
Leu Gly Ala Ala Leu
ATP binding domain
Pfu polymerase
Extension.
Transfection
And expression
Pfu polymerase
Extension, nick synthesis.
Which amino acid to substitute?
Alanine, which has a small inert side chain is typically used for aa substitution in
Site-directed-mutagenesis. However, one must take into account structure in
Cases of amino acids so as not to disturb secondary structure of the protein and
Most closely mimic the original amino acid which has been substituted.
END