Download Molecular Diagnosis I: Methods in Molecular Medicine 张咸宁

Molecular Diagnosis I: Methods in
Molecular Medicine
张咸宁
[email protected]
Tel：13105819271; 88208367
Office: A705, Research Building
2012/09
Trends of Medicine in 21th Century
• Application of genomic knowledge &
technology will significantly improve
the diagnosis and treatment of the
disease
– Treatment afterward Predictive & Preventive
– General Personalized
– Molecular epidemiology: Sub-population
– Rare diseases  Common diseases
Why Clone Human Genes?
•To define inherited genetic mutations
•Develop diagnostic reagents (pre-natal testing)
•Develop targeted therapies
•To isolate functional/normal genes
•Prepare normal protein products (Factor VIII)
•Therapeutic use of proteins normally produced at low
levels
•Vaccine development (avoid attenuated organisms)
•To isolate genes with somatic mutations
•Develop diagnostic reagents
•Target therapy to altered gene product
•To define and understand cellular pathways and
circuits
•Signal transduction, immunity, development, cell cycle
and apoptosis
The basic tools of gene exploration
• Restriction-enzyme analysis: “molecular scalpels”
• Recombinant DNA technique:
• Blotting technique:
1. Southern Blot: DNA hybridization
2. Northern Blot: RNA hybridization
3. Western Blot: Protein hybridization
• DNA sequencing: observe the DNA sequence
• Solid-phase synthesis of nucleic acids: synthesize nucleic
acids sequence de novo
• PCR (polymerase chain reaction): amplify DNA
billionfold
Overview Molecular Diagnostics and Therapeutics
• Identify Changes in DNA Sequence
• Identify Changes in Gene Expression
– Individual Gene
– Global Gene Expression Profiling
• Develop Disease Targeted Therapies
• Impact of the Human Genome Project
Human genome DNA Extraction
• Venous blood(5 ml) → WBC → lysing
buffer (SDS) and protease K →
centrifugation, collect supernatant →
phenol → centrifugation, collect
supernant → RNase A and T1 → phenol
→ centrifugation, collect supernatant
→ potassium acetate and EtOH
(absolute) → genomic DNA appears
(like cotton fiber)
Restriction-enzyme analysis: “molecular
scalpels”—Restriction endonucleases split
DNA into specific fragment
• RE: recognize specific base sequences in doublehelical DNA and cleave at specific places.
• Types of RE: >100
Nomenclature of RE: A 3-letter abbreviation
for the host organism (italic!) + a strain
designation + a roman numeral
• E.g., EcoRI: (Escherichia coli)
sticky end
blunt end
Restriction fragments can be separated
by gel electrophoresis and visualized
• 1, 4,7: fX-174 RF DNA-Hind II digest; 2, 5, 8: lambda
DNA-Hind III digest; 3, 6: fX-174 RF DNA-Hae III
digest. Markers were separated for 30 min at 200 V in a
1% agarose gel, then stained with ethidium bromide(EB).
Electrophoresis
Horizontal electrophoresis system
Vertical electrophoresis system
Range of Separation in Cells Containing
Different Amounts of Standard Low-EEO
(electroendo-osmosis) Agarose:
Agarose Concentration Range of Separation of
in Gel (% [w/v])
Linear DNA Molecules (kb)
0.3
5 ~ 60
0.6
1 ~ 20
0.7
0.8 ~ 10
0.9
0.5 ~ 7
1.2
0.4 ~ 6
1.5
0.2 ~ 3
2.0
0.1 ~ 2
Effective Range of Separation of DNAs in
Polyacrylamide gel (PAGE)
Concentration of
Effective Range of
Separation
Acrylamide Monomer (%)
(bp)
3.5
1000 ~ 2000
5.0
80 ~ 500
8.0
60 ~ 400
12.0
40 ~ 200
15.0
25 ~ 150
20.0
6 ~ 100
Pulsed-field Gel Electrophoresis (PFGE)
Resolution: 20 kb ~100 mb
E.g., Gene Navigator Pulsed Field System
(GE,USA)
Analyzing specific nucleic acids in complex mixtures
The Southern Blot- Edwin Southern
• Digest the total DNA of an organism with a RE
• fractionate by size
• Identify sequence of interest using a labeled probe
Southern Blot: DNA hybridization
Southern Blot: DNA hybridization
Southern Blot: DNA hybridization
Southern blots are used for diagnostic
procedures
•Large deletions
•Point mutations which alter a RE site
•Chromosomal translocations
•Gene amplification
Northern blot: RNA hybridization
• Expression of a specific gene-changes from
tissue to tissue
• The relative size of the mRNA transcript
• Relative levels of RNA in different samples
Northern Blot analysis reveals increased
expression of β-globin mRNA in differentiated
erythroleukemia cells. UN: uninduced cells
Western Blot: Protein hybridization
(A) Blot using
the Dy4/6D3
Ab, which is
specific for the
dystrophin rod
domian.
(B) Blot using
the Dy6/C5 Ab,
which is
specific for the
C-terminal
region of
dystrophin.
DNA sequencing : Sanger (dideoxy) method,
using flurescent tagged ddNTPs.
The 96-capillary 3730xl DNA Analyzer
(ABI,USA) is the Gold Standard for high
throughput genetic analysis
Frederick Sanger (1918-) : Nobel Prize
Winner 2 times:
Pr. Sequencing/1958; DNA Sequencing/1980
DNA probes and genes can be
synthesized by automated solid-phase
methods: ABI 310 DNA Synthesizer
Millions of the target sequences can be
readily obtained by PCR if the flanking
sequences of the target are known.
3 steps:
• Denature
• Renature (annealing)
• Extension
ABI 9700 PCR System
PCR amplification of DNA
Cycle
3
4
5
10
20
30
ds copies
2
4
8
256
262,144
268,435,456
PCR is used in many diagnostic tests and
forensic tests
•Point mutations can be detected by
incorporating them into the primers
•PCR product can be sequenced
•Sizing of repeated regions-microsatellite
•Detecting infectious diseases (viral genomes)
PCR is powerful in lots of biomedical
fields! Mullis K (1993 NP )
RT: Formation of a cDNA duplex
Quantitative RT-PCR
Measures fluorescence generated by incorporation of
a tagged nucleotide (SURF-4 mRNA)
The expression levels of thousands of genes
can be simultaneously analyzed using DNA
microarrays
• The level at which a gene is expressed,as
indicated by mRNA quantities,can vary
widely,ranging from no expression to
hundreds of mRNA copies per cell.Geneexpression patterns vary from cell type to
cell type.
• Even within the same cell, gene-expression
levels may vary as the cell responds to
changes in physiological circumstances.
DNA microarrays (gene chips)
Cells, cell lines, tumor specimens etc.
Genes
Recombinant DNA technology
• Recombinant DNA:Any DNA
molecule formed in vitro by joining
DNA fragments from different
sources. Commonly produced by
cutting DNA molecules with
restriction enzymes and then joining
the resulting fragments from
different sources with DNA ligase.
Recombinant DNA technology: REs and DNA
ligase (“molecular paste”) are key tools
Vectors: Plasmid, λ phage,…
• Plasmid: Small,circular extrachromosomal
DNA molecule capable of autonomous
replication in a cell.
Use of recombinant DNA technology to
produce medicine
•Production of vaccines
Yeast cells
Can be grown to high density in a fermentor
Acknowledge（PPT特别鸣谢！）
• UCLA David Geffen School of Medicine
• www.medsch.ucla.edu/ANGEL/
• Prof. Gasson JC (UCLA Jonsson
Comprehensive Cancer Center ), et al.