Molecular Diagnosis I: Methods in Molecular Medicine

Molecular Diagnosis I: Methods in Molecular Diagnosis I: Methods in Molecular MedicineMolecular Medicine

张咸宁zhangxianning@zju.edu.cn

Tel ： 13105819271; 88208367 Office: A705, Research Building

2012/09

Trends of Medicine in 21th CenturyTrends of Medicine in 21th Century

• Application of genomic knowledge & Application of genomic knowledge & technology will significantly improve the technology will significantly improve the diagnosis and treatment of the diseasediagnosis and treatment of the disease

– Treatment afterward Predictive & Preventive

– Molecular epidemiology: Sub-population

– Rare diseases Common diseases

– General Personalized

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

double-helical 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: X-174 RF DNA-Hind II digest; 2, 5, 8: lambda DNA-Hind III digest; 3, 6: X-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 ds copies 3 2 4 4 5 810 25620 262,14430 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.Gene-expression 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)

Genes

Cells, cell lines, tumor specimens etc.

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. Prof. Gasson JC ((UCLA Jonsson Comprehensive Cancer Center ), et al.), et al.