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Molecular Medicine 分子醫學
高英賢 義大醫院醫學研究部
參考書籍: Textbook of Molecular Diagnosis in Medicine, 3rd edition (吳俊忠/李宏謨/孫光蕙/趙崇義主編)
Lecture Contents 周數 日期 課程主題 授課老師 備註
1 9/16 Basic technology for molecular medicine 義大醫院 醫學研究部 高英賢博士
4 hrs (考題25%)
3 9/30 1. Biochips & bioinformatics for molecular
diagnosis 2. Molecular diagnostics for cancer target therapy
義大醫院 醫學研究部 林宇駿博士
4 hrs (考題25%)
5 10/14 1. Molecular diagnosis of infectious diseases 2. Molecular diagnosis of hematological
malignancies and anemia
義大醫院 醫學檢驗部 王秋惠主任
4 hrs (考題25%)
7 10/28 Molecular diagnosis of genetic disorders 高英賢博士 4 hrs (考題25%)
9 11/11 Mid-term exam (筆試) 高英賢博士 11 11/25 1. The principle for biomarker detection and
examination 2. Pharmacogenomics in molecular diagnosis
義大醫院 醫學研究部 陳俊杰博士
4 hrs
13 12/9 Molecular Paternity test & quality assurance in
molecular diagnostics 高英賢博士 4 hrs
15 12/23 1. Molecular HLA typing 2. Molecular diagnosis of immune disease:
Hypersensivity & Immunodeficiency
義大醫院 醫學研究部 戴宗玄博士
4 hrs
17 1/6 Seminar (口頭報告; 12/31前繳書面報告) 高英賢博士 4 hrs
18 1/13 Final exam 高英賢博士
評分標準: 出席 10%; 期中考30%; 書面報告30%; 期末考(口頭報告)30%
Principal Discoveries in Molecular Biology (I)
Year Discovery
1949 Characterization of sickle cell anemia as a molecular disease
1953 Discovery of the DAN double helix
1958 Isolation of DNA polymerase
1960 First hybridization techniques
1969 In situ hybridization
1970 Discovery of restriction enzymes and reverse transcriptase
1975 Southern blotting
1977 DNA sequencing
1983 First synthesis of oligonucleotides
1985 Invention of PCR, Matrix-assisted laser desorption ionization (MALDI)
1986 Development of fluorescent in situ hybridization (FISH)
1988 Discovery of the thermostable DNA polymerase – PCR optimization
1992 Conception of real time PCR
Principal Discoveries in Molecular Biology (II)
Year Discovery
1993 Discovery of structure-specific endonucleases for cleaveage assays
1996 First application of DNA microarrays
2001 First draft version of the human genome sequence
2001 Application of protein profiling in human diseases (proteomics)
2003 Completion of human genome project
… Post-genomic era begins….
… Bioinformatics
… Translational research
… Next generation sequencing (NGS)
分子生物 相關資料
電腦運算 +
Bioinformatics
Major Trends in Biomedical Sciences
Genomics
Proteomics
Metabolomics
Transcriptomics
Epigenomics
Epigenetic modifications are chemical modifications to the genome
that play a role in development, aging, health, and disease, and are
therefore targets for therapeutic interventions.
Human Microbiomics To characterize the microbial communities found at several different sites on the
human body, and to analyze the role of these microbes in human health and
disease.
person A Person B
Most likely
Drug acts on therapy
Most likely
Drug has no efficacy
Drug
treatment
Choose other
drugs
Genotype Screening
Personalized Medicine
Whole genome sequencing
Development of personalized medicine is under way!
Personal prescription
Pharmacogenomics
Preventive treatment
Next Generation Sequencing (NGS) Biotechnology
第一單元 分子檢驗技術
• 第一章 分子檢驗基本技術
(Basic Technology for Molecular Diagnosis)
MOLECULAR BIOLOGY – Molecular biology techniques
1866 1910
1944 1953
Mendel
Morgan
Watson & Crick Avery, MacLeod & McCarty
human DNA:
3 200 000 000 letters
Approx 25,000 genes
filling 150x
MOLECULAR BIOLOGY – Molecular biology techniques
Human genome sequence
NOT BAD WORK
INSIDE 150 YEARS !!!!
1977
Sanger
2004
Table 8-3 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
START STOP
A U G G C A A U C A A G U G C U A A
RNA T A C C G T T A G T T C A C G A T T
A T G G C A A T C A A G T G C T A A
GTTTATTGCATTCTTCTGTGAAAAGAAGCTGTTCACAGAATGATTCTGAAGAACCAACTT
TGTCCTTAACTAGCTCTTTTGGGACAATTCTGAGGAAATGTTCTAGAAATGAAACATGTT
CTAATAATACAGTAATCTCTCAGGATCTTGATTATAAAGAAGCAAAATGTAATAAGGAAA
AACTACAGTTATTTATTACCCCAGAAGCTGATTCTCTGTCATGCCTGCAGGAAGGACAGT
GTGAAAATGATCCAAAAAGCAAAAAAGTTTCAGATATAAAAGAAGAGGTCTTGGCTGCAG
CATGTCACCCAGTACAACATTCAAAAGTGGAATACAGTGATACTGACTTTCAATCCCAGA
AAAGTCTTTTATATGATCATGAAAATGCCAGCACTCTTATTTTAACTCCTACTTCCAAGG
ATGTTCTGTCAAACCTAGTCATGATTTCTAGAGGCAAAGAATCATACAAAATGTCAGACA
AGCTCAAAGGTAACAATTATGAATCTGATGTTGAATTAACCAAAAATATTCCCATGGAAA
AGAATCAAGATGTATGTGCTTTAAATGAAAATTATAAAAACGTTGAGCTGTTGCCACCTG
AAAAATACATGAGAGTAGCATCACCTTCAAGAAAGGTACAATTCAACCAAAACACAAATC
TAAGAGTAATCCAAAAAAATCAAGAAGAAACTACTTCAATTTCAAAAATAACTGTCAATC
CAGACTCTGAAGAACTTTTCTCAGACAATGAGAATAATTTTGTCTTCCAAGTAGCTAATG
AAAGGAATAATCTTGCTTTAGGAAATACTAAGGAACTTCATGAAACAGACTTGACTTGTG
TAAACGAACCCATTTTCAAGAACTCTACCATGGTTTTATATGGAGACACAGGTGATAAAC
AAGCAACCCAAGTGTCAATTAAAAAAGATTTGGTTTATGTTCTTGCAGAGGAGAACAAAA
ATAGTGTAAAGCAGCATATAAAAATGACTCTAGGTCAAGATTTAAAATCGGACATCTCCT
TGAATATAGATAAAATACCAGAAAAAAATAATGATTACATGAACAAATGGGCAGGACTCT
TAGGTCCAATTTCAAATCACAGTTTTGGAGGTAGCTTCAGAACAGCTTCAAATAAGGAAA
TCAAGCTCTCTGAACATAACATTAAGAAGAGCAAAATGTTCTTCAAAGATATTGAAGAAC
AATATCCTACTAGTTTAGCTTGTGTTGAAATTGTAAATACCTTGGCATTAGATAATCAAA
AGAAACTGAGCAAGCCTCAGTCAATTAATACTGTATCTGCACATTTACAGAGTAGTGTAG
TTGTTTCTGATTGTAAAAATAGTCATATAACCCCTCAGATGTTATTTTCCAAGCAGGATT
TTAATTCAAACCATAATTTAACACCTAGCCAAAAGGCAGAAATTACAGAACTTTCTACTA
TATTAGAAGAATCAGGAAGTCAGTTTGAATTTACTCAGTTTAGAAAACCAAGCTACATAT
TGCAGAAGAGTACATTTGAAGTGCCTGAAAACCAGATGACTATCTTAAAGACCACTTCTG
AGGAATGCAGAGATGCTGATCTTCATGTCATAATGAATGCCCCATCGATTGGTCAGGTAG
ACAGCAGCAAGCAATTTGAAGGTACAGTTGAAATTAAACGGAAGTTTGCTGGCCTGTTGA
AAAATGACTGTAACAAAAGTGCTTCTGGTTATTTAACAGATGAAAATGAAGTGGGGTTTA
GGGGCTTTTATTCTGCTCATGGCACAAAACTGAATGTTTCTACTGAAGCTCTGCAAAAAG
CTGTGAAACTGTTTAGTGATATTGAGAATATTAGTGAGGAAACTTCTGCAGAGGTACATC
CAATAAGTTTATCTTCAAGTAAATGTCATGATTCTGTTGTTTCAATGTTTAAGATAGAAA
ATCATAATGATAAAACTGTAAGTGAAAAAAATAATAAATGCCAACTGATATTACAAAATA
ATATTGAAATGACTACTGGCACTTTTGTTGAAGAAATTACTGAAAATTACAAGAGAAATA
CTGAAAATGAAGATAACAAATATACTGCTGCCAGTAGAAATTCTCATAACTTAGAATTTG
ATGGCAGTGATTCAAGTAAAAATGATACTGTTTGTATTCATAAAGATGAAACGGACTTGC
TATTTACTGATCAGCACAACATATGTCTTAAATTATCTGGCCAGTTTATGAAGGAGGGAA
ACACTCAGATTAAAGAAGATTTGTCAGATTTAACTTTTTTGGAAGTTGCGAAAGCTCAAG
MOLECULAR BIOLOGY – Molecular biology techniques
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
MOLECULAR BIOLOGY – Molecular biology techniques
ISOLATION OF DNA
High MW Genomic DNA Isolation
Typical Procedure
1 Cell Lysis
– 0.5% SDS + proteinase
K (55oC several hours)
2 Phenol Extraction
– Gentle rocking several
hours
Phenol Extraction • mix sample with equal volume
of sat. phenol soln
• retain aqueous phase
• optional chloroform/isoamyl
alcohol extraction(s)
aqueous phase
(nucleic acids)
phenolic phase
(proteins)
MOLECULAR BIOLOGY – Molecular biology techniques
ORGANIC PHASE SEPARATION
High MW Genomic DNA Isolation
Typical Procedure
1 Cell Lysis
– 0.5% SDS + proteinase K
(55oC several hours)
2 Phenol Extraction
– gentle rocking several
hours
3 Ethanol/ salt Precipitation
EtOH Precipitation • 2-2.5 volumes EtOH, -20oC
• high salt, pH 5-5.5
• centrifuge or ‘spool’ out
MOLECULAR BIOLOGY – Molecular biology techniques
4 RNAse followed by
proteinase K
5 Repeat Phenol Extraction
and EtOH ppt
PLASMID DNA
MOLECULAR BIOLOGY – Molecular biology techniques
Natural Bacterial Transformation/ conjugation
Also possible to experimentally
‘transform’ plasmid vectors into bacteria -
see later S. Pneumoniae ‘transforming’ DNA is a
plasmid
PLASMID DNA ISOLATION Alkaline lysis denaturation/ renaturation protocol
MOLECULAR BIOLOGY – Molecular biology techniques
Protein denaturation (SDS)
Single stranded plasmid DNA
Single stranded genomic DNA
Bacteria lysed in
SDS + strong NaOH
buffer
DENATURATION
Small multi-copy plasmid DNA
quickly re-anneals in solution
Large single copy genomic
DNA fails to re-anneal and
forms precipitate with proteins
Potassium
acetate
pH
NEUTRALISATION
SEDIMENTATION
Centrifugation
Aqueous (double
stranded plasmid
DNA)
Pellet (proteins
and genomic
DNA)
MOLECULAR BIOLOGY – Molecular biology techniques
PLASMID DNA ISOLATION Aqueous (double
stranded plasmid
DNA)
Pellet (proteins
and genomic
DNA)
1. Phenol/ CHCl3 extraction & Ethanol/ Salt precipitation
or 2. Solid phase/ silica extraction ‘miniprep’
Quick relatively pure double stranded
plasmid DNA
In presence of alkaline
chaotropic salts, denatured
plasmid DNA binds to silica
beads in the column
Wash buffers used to remove impurities
&
DNA eluted (and re-natured in H2O)
Centrifugation steps
MOLECULAR BIOLOGY – Molecular biology techniques
PLASMID DNA ISOLATION Aqueous (double
stranded plasmid
DNA)
Pellet (proteins
and genomic
DNA)
or 3. Anion exchange column-based chromatography
Altering the pH and ionic conditions removes impurities
leading to high [salt] elution and EtOH or isopropanol
precipitation
Extremley pure double stranded plasmid
DNA
Isolation of RNA Special Considerations
• RNAse inhibitors!
• extraction in guanidine salts
• phenol extractions at pH 5-6
• (pH 8 for DNA)
• selective precipitation of high MW
forms (rRNA, mRNA) with LiCl
• oligo-dT column for mRNA’s
• treatment with RNase-free DNase
MOLECULAR BIOLOGY – Molecular biology techniques
Guanidinium thiocyanate
Using UV spectroscopy to analyze DNA/ RNA • Nucleic acids absorbs UV light with a major peak at 260nm (max)
Ab
so
rba
nce
Wave Length ()
MOLECULAR BIOLOGY – Molecular biology techniques
• Detection
• Quantitation
• Assessment of purity
• Absorbance extinction coefficients () vary depending on the nucleic acid structure
A260
Isolated
nucleotides
ss RNA/ DNA
= 25
ds DNA
= 20
• A260 / A280 ratio indicates sample purity
Pure RNA = 2.0
Pure DNA = 1.8
Beer-Lambert equation
A = cl
RNA Integrity Number (RIN) Standardization of RNA Quality Control
Agilent 2100 bioanalyzer
Increasing degradation of RNA
So now we have isolated DNA ... but it is still too long to work with:
How to fragment it?
- mechanical shearing
(no control)
or ...
MOLECULAR BIOLOGY – Molecular biology techniques
MOLECULAR SCISSORS - TYPE II RESTRICTION ENDONUCLEASES
Hamilton Othanel Smith 1968
cohesive ends
MOLECULAR BIOLOGY – Molecular biology techniques
SPECIFIC CUT SPECIFIC JOINING (LIGATION)
Ability to join two foreign pieces of DNA together
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
MOLECULAR BIOLOGY – Molecular biology techniques
BLUNT
END
CO
HE
SIV
E E
ND
S
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 (RE) 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
RECOMBINANT DNA TECHNOLGY
MOLECULAR BIOLOGY – Molecular biology techniques
The plasmid as DNA ‘vector’ (vehicle)
Possible to insert ‘interesting’
DNA’s into a plasmid using
restriction endonucleases
RECOMBINANT DNA TECHNOLGY
MOLECULAR BIOLOGY – Molecular biology techniques
The plasmid as DNA ‘vector’ (vehicle)
The recombinant plasmid containing the
‘interesting’ DNA sequence can now be
propagated/ amplified by experimentally
transforming the recombinant plasmid
into bacteria and allowing these bacteria
to multiply and produce more
recombinant plasmid
Specialized strains of bacteria can be permeablised
by electroporation of heat shock
Therefore specific DNA fragments can
be selectively propagated i.e. cloned
RECOMBINANT DNA TECHNOLGY
MOLECULAR BIOLOGY – Molecular biology techniques
Specialized plasmid cloning vectors
Muliple Cloning Site (MCS) contains many
restriction sites to maximize target DNA
cloning potential
Plasmids contain genes that confer antibiotic
resistance so that only successfully
transformed bacteria are propagated
3 Why not clone whole genomes?
MOLECULAR BIOLOGY – Molecular biology techniques
Each bacterial colony represents an amplified
clone containing a recombinant plasmid
harbouring a distinct region of the genome
i.e. together they represent a ‘Genomic
DNA Library’
Also possible to do this using cDNA copies of transcribed mRNAs resulting a
‘cDNA Gene Expression Library’
Hybridization
to filter
cloning
GENOMIC or cDNA EXPRESSION LIBRARY
MOLECULAR BIOLOGY – Molecular biology techniques
isolate
DNA
.
genomic library
hybridisation
restriction
digestion
MOLECULAR BIOLOGY – Molecular biology techniques
sub-clone
experimentation
How to see genes ...
... where in
tissues?
... where on
chromosomes?
How to see specific
forms of genes?
How to clone
and amplify
genes?
southern blot
RFLP
in situ hybridization recombinant DNA
MOLECULAR BIOLOGY – Molecular biology techniques
genomic libraries
MOLECULAR BIOLOGY – Molecular biology techniques
Bacteriophage Lambda
vectors
phage linear DNA genome
Non-essential region allowing that can be
substituted by DNA to be cloned (approx 20Kb)
cos cos
Cosmids, phosmids, BACs and YACs to clone larger DNA fragments
DNA ISOLATION AMPLIFICATION
How to visualize DNA?
GEL ELECTROPHORESIS NUCLEIC ACID HYBRIDIZATION
size distinction sequence distinction
MOLECULAR BIOLOGY – Molecular biology techniques
+
- - -
GEL ELECTROPHORESIS Fragmented DNA
MOLECULAR BIOLOGY – Molecular biology techniques
D-galactose 3,6-anhydro
L-galactose n
agarose
Ethidium Bromide SYBR® Safe on blue light
MOLECULAR BIOLOGY – Molecular biology techniques
23 kb
9,5 kb
Genomic DNA on gel:
MOLECULAR BIOLOGY – Molecular biology techniques
Plasmid DNA on gel:
MOLECULAR BIOLOGY – Molecular biology techniques
NUCLEIC ACID HYBRIDIZATION
Fluorescence In Situ Hybridization
(FISH)
labeled probe
MOLECULAR BIOLOGY – Molecular biology techniques
Metaphase spread chromosomes
on a slide
Biotin-11-dUTP
32P
Radioactive labeling
Probe labeling by incorporation of modified (d)NTPs
AUTORADIOGRAPHY
Streptavidin
Y
anti-DIG
antibody
(DIG)
Fluorophores
conjugation
Enzymes alkaline phosphatase
horseradish peroxidase
chemiluminiscence
MOLECULAR BIOLOGY – Molecular biology techniques
DIG
DIG
DIG
DIG
DIG
DIG
DIG
DIG
DIG
HRP substrate HRP
substrate HRP substrate HRP
substrate HRP
substrate
MOLECULAR BIOLOGY – Molecular biology techniques
DNA denaturation
Melting (denaturation) temperature
depends on these major factors:
- GC content (and therefore AT content)
- sequence length
- gaps in the annealed strands
- salt concentration
- pH
- organic solvents (DMSO, formamide...)
GC rich
GC rich
AT rich
Temp Temp
MOLECULAR BIOLOGY – Molecular biology techniques
原位螢光雜交法 (Fluorescence in situ Hybridization; FISH)
• 原位螢光雜交法(FISH)是利用螢光探針與細胞內染色體中 DNA 的序列接合,在螢光顯微鏡下觀察偵測細胞內核酸的技術,可辨認特定 DNA 序列是否存在染色體中。
• 此技術應用在遺傳性基因檢測、藥物篩檢、種別判定,也用於檢測特殊 mRNA 存在組織中的位置,及確定基因在細胞及組織中表現的狀態。
等位基因特異性寡核酸雜交 Allele-specific Oligonucleotide
Hybridization (ASO)
• 目前,等位基因特異性寡核酸雜交反應已廣泛應用
於癌症、疾病與親子鑑定等檢測及研究上,各種常見
的 SNP 基因鑑定方法大多已整合雜交與聚合連鎖反
應(polymerase chain reaction; PCR)。
MOLECULAR BIOLOGY – Molecular biology techniques
CHROMOSOME PAINTING – MULTI COLOR FISH
MOLECULAR BIOLOGY – Molecular biology techniques
Particularly useful when diagnosing chromsomal abnormalities in certain forms of
cancer (region specific barcoding on left and whole chromsosome paints on right)
Translocated chromsome segment
Where in organism is the gene expressed?
Detection of mRNA by in situ hybridization:
MOLECULAR BIOLOGY – Molecular biology techniques
Adaptation of DNA FISH protocol (removal of genomic DNA by predigestion with
DNase and use of labeled RNA probes to detect expressed transcripts)
MOLECULAR BIOLOGY – Molecular biology techniques
How to analyze specific form of genes in genomic DNA?
e.g. successful intergration of a transgene into the genome of a
transgeneic animal (mouse)
The Southern Blot- Edwin Southern
• Digest the total DNA of an organism with a RE
• Fractionated by size
• Identify sequence of interest using a labeled probe
Analyzing specific nucleic acids in complex mixtures
Electrophoresis
Horizontal 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
脈衝式電泳分析 Pulsed Field Gel Electrophoresis (PFGE)
– 脈衝式電泳儀器設計
– 脈衝式電泳分析法之設計原理
PFGE 操作流程示意圖
Pulsed-field Gel Electrophoresis (PFGE)
Resolution: 20 kb ~100 mb
E.g., Gene Navigator Pulsed Field System (GE,USA)
Figure 8-38 (part 1 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
Southern blot – transfer of DNA to membrane
fragmented
DNA
e.g. fragmentation by restriction
endonucleases
Figure 8-38 (part 2 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
Figure 8-38 (part 3 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
Labeled probe has sequence
homology to DNA of interest e.g.
the hopefully integrated
transgene
Figure 8-38 (part 4 of 4) Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
e.g. bands reveal
integrated transgenes
and size shows whether
integration was correct
Figure 8-38 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Molecular biology techniques
SOUTHERN BLOT combines DNA fragmentation,
gel electrophoresis and hybridization
to analyze specific DNA sequences
Same procedure blotting RNA used to
confirm gene mRNA expression called
NORTHERN BLOTTING
Similar principle used to blot proteins that are then
detected by specific antibodies - WESTERN
BLOTTING
Southern Blot: DNA hybridization
Southern Blot: DNA hybridization
•Large deletions
•Point mutations which alter a RE site
•Chromosomal translocations
•Gene amplification
Southern blots are used for diagnostic procedures
• 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: RNA hybridization
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.
Frederick Sanger (1918-) : Nobel Prize Winner 2 times:
Pr. Sequencing/1958; DNA Sequencing/1980
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
聚合酶連鎖反應 Polymerase Chain Reaction (PCR)
PCR 技術非常簡單而直接,其原理完全仿照自然界 DNA
合成的步驟,只是加以自動化而已。
三步驟:
DNA 雙股分離(Denaturation)
引子結合(Annealing)
引子延伸(Extension)
PCR is powerful in lots of biomedical fields! Kary B. Mullis (1993 NP )
• The Nobel Prize in Chemistry 1993 was awarded "for contributions to the developments of methods within DNA-based chemistry“ jointly with one half to Kary B. Mullis "for his invention of the polymerase chain reaction (PCR) method" and with one half to Michael Smith "for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies".
K.B. Mullis
Cetus Corp., 1983 M. Smith
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
•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 used in many diagnostic tests and forensic tests
– Multiplex PCR: 一次放入多對引子進行PCR反應,同時放大檢測多組基因訊號。
– 巢式聚合酶連鎖反應 (Nested PCR) 改善聚合酶連鎖反應 引子之專一性
– 反轉錄—聚合酶連鎖反應 (Reverse transcriptase PCR)
– 序列特異聚合酶連鎖反應(Sequence-specific PCR)
– 即時聚合酶連鎖反應(Real-time PCR; quantitative PCR or qPCR)
RT: Formation of a cDNA duplex
Quantitative RT-PCR
Measures fluorescence generated by incorporation of a tagged nucleotide (SURF-4 mRNA)
限制片段長度多形性 Restriction Fragment Length Polymorphism
(RFLP)
• 1980 年代,很快的被全球用來當作 DNA標準測試技術,也是最早被用來作為人類遺傳圖譜比對的工具。
• RFLP 目前常見應用於遺傳指紋(genetic finger printing)、親子鑑定與遺傳疾病分析等。
增幅限制切位點 Amplified created restriction site (ACRS)
原理:
首先要知道欲分析基因的突變位點,之後在引子上設計帶有限制切
位的部分序列,在 PCR 的時候此引子會增幅出具有正常的限制酶
切位序列,或因突變位點導致增幅出具有突變的限制酶切位序列。
在 PCR 增幅反應後,進行限制酶切割反應。此時,具有正常限制
酶切位的片段就會被切割,突變的片段則否,在瓊脂凝膠電泳反應
後,可以發現正常與突變的基因片段大小不同來判斷該位點是否產
生突變。
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.
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).
Restriction Fragment Length Polymorphism (RFLP)
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATTCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAA’3
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATGCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’3
SNP
EcoRI
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTG AATTCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTAA GACGCTACAA’3
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATGCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’3
Restriction digestion by EcoRI
MOLECULAR BIOLOGY – Molecular biology techniques
RFLP
MOLECULAR BIOLOGY – Molecular biology techniques
Diagnosis of Genetic Diseases by RFLP
MOLECULAR BIOLOGY – Molecular biology techniques
單股結構多形性 Single Strand Conformation Polymorphism
(SSCP) 基本原理:
• 以 PCR 來擴增目標DNA
• 將特異性的 PCR 擴增產物在高溫下使DNA 變性(denature)
• 快速置於冰上使其在單股結構狀態下回復(renature),成為具有一定空間結構的單股 DNA 分子
• 將適量的單股 DNA 進行非變性聚丙烯醯胺凝膠電泳(non-denatured PAGE)
• 最後通過放射性顯影(Autography)、銀染(Silver stain) 或溴化乙錠(Ethidium bromide; EtBr)顯色分析結果。
• 若發現單股 DNA條帶(band)移動率與正常對照的相對位置發生改變,就可以判定該股結構發生改變,進而推斷該DNA片段中有鹼基突變。
單股結構多形性 Single Strand Conformation Polymorphism
(SSCP)
增幅阻礙突變系統 Amplified Refractory Mutation System (ARMS)
原理:
在 PCR 的引子設計上能增幅與區別單一點突變的引
子對,引子對之一股能夠完全結合標的基因,引子對
的另一股則需特別設計,依據已知 DNA 序列上正常
型或是突變型來設計,引子僅 3‘ 末端的鹼基不同,分
別會與正常型或是突變型的基因片段來結合。
突變分辨聚合連鎖反應 Mutation-specific PCR (MS-PCR)
化學切割錯誤的鹼基 Chemical cleavage of mismatched duplexes (CCM)
基本步驟: (1)利用 PCR將欲分析之 DNA 片段放大。
(2)將樣本中可能出現變異的 DNA 片段和正常型(或野生型)的 DNA 片段混合,加熱至 95°C使 DNA 解離成單股結構後,再將溫度降到 65°C,使單股 DNA 互相結合形成異股DNA(heteroduplex) 。
(3)異股 DNA 與兩種化學藥劑反應,這些化學藥劑特別針對配對錯誤(mismatch)的鹼基進行修飾,其中 Hydroxylamine 修飾配對錯誤鹼基中的胞嘧啶(Cytosine),過錳酸鉀(KMnO4)則會修飾配對錯誤鹼基中的胸腺嘧啶(Thymine) 。
(4)後續加入的 Piperidine 會在 DNA 上被修飾的胞嘧啶及胸腺嘧啶位置進行切割。
(5)切割後的反應物經由電泳分離,可以分析出特定片段內是否含有突變。
變性梯度膠體電泳法 Denaturing gradient gel electrophoresis
(DGGE)
變性梯度膠體電泳法(DGGE)是一種分子指
紋技術,可以藉由分離 PCR 產物,區別 PCR
擴增的區域中是否有變異(variations)或突
變(mutations)產生。
應用核酸序列的放大反應 Nucleic acid sequence based amplification
(NASBA)
• NASBA是一項連續恆溫(isothermal)的核酸擴增技術,用於
RNA 的放大與偵測。此技術使用到:
(1)三種酵素
(2)dNTP(deoxynucleotide triphosphate;去氧核糖核)及 NTP(nucleotide triphosphate;核酸)
(3)兩條引子(primers)
轉錄介導的擴增法 Transcription mediated amplification
(TMA)
– 三步驟:
• 檢體製備
• 擴增
• 偵測
連接酶連鎖反應 Ligase chain reaction (LCR)
– 連接酶連鎖反應的原理當兩條專一性的相鄰人工合成的寡核苷酸引子與標的 DNA 的其中一股互相配對時,再利用連接酶將此兩條相鄰的寡核苷酸連接起來。
– 連接酶連鎖反應的重要因素
• 連接酶連鎖反應引子的設計:
• 連接酶連鎖反應的反應條件:
– 連接酶連鎖反應產物的偵測法
• 早期~利用 32P 同位素標記在上游引子的 3‘ 端,
再藉由變性膠體電泳分離後,以自動同位素照相術偵測連接酶連鎖反應產物。
應用非同位素的偵測方式,使用螢光標記的引子,配合使用螢光 DNA 定序儀與掃描儀(GENESCANNER, Applied Biosystems)
– 連接酶連鎖反應的應用
• 偵測遺傳疾病
• 細菌和病毒感染之檢測
分枝 DNA 訊號放大技術 Branched DNA (b-DNA) amplification
此技術是以訊號的擴增為設計原理,欲偵測的核酸序列並未被複製,而是利用偵測訊號的擴增,來提高反應的靈敏度。由於不涉及核酸序列的放大,因此,實驗中由汙染所產生的非專一性核酸序列放大的機會,遠比 PCR 反應來得低。而且此方法也比 PCR 反應更能忍受目標序列變異所造成的影響,更直接的偵測目標物、檢體製備更簡易,以及檢體與檢體間的差異性更低。
分枝 DNA 訊號放大技術 Branched DNA (b-DNA) amplification
DNA 甲基化分析技術
• Epigenetics
DNA 甲基化分析技術
基本原理~
所選的兩個同切位限制酶,它們所辨認的切割位置如果被甲基化時,其中一個限制酶無法切割此甲基化的位置,而另一個限制酶則不受甲基化影響。因此在進行南方墨點分析法時,就可以很容易的根據片段大小差異而區分此區域是否發生甲基化。此方法因為操作相當簡單、成本低且判讀容易,所以特別適合使用於大量檢體的篩檢。
DNMT: DNA methyltransferase
重亞硫酸鹽法 (Bisulfite conversion method)
– 重亞硫酸鈉會使單股 DNA 上的胞嘧啶脫氨,在酸性 pH值下會形成 5,6-雙氫胞嘧啶-6-鈉磺酸鹽(5,6-dihydrocytosine-6-sodium sulphonate),是此反應的間接產物,然後再轉換為鹼性環境,導致重亞硫酸鈉降解而使得間接產物轉變為尿嘧啶(uracil)。
– 5-甲基胞嘧啶(5-methylcytosine)也可能會進行此反應(脫氨成為胸腺嘧啶),但是此反應速率很慢且會抑制終產物的形成。所以最後其鹼基型態仍為胞嘧啶,並不會受到改變,所以經重亞硫酸鈉處理後,可以利用 PCR 配合限制酶切割的方式來分析 DNA 甲基化的情形。
重亞硫酸鹽法 (Bisulfite conversion method)
•甲基化專一性 PCR 反應(Methylation Specific PCR; MS-PCR)
•即時定量 PCR(Real-Time PCR, Methy Light)
•甲基化依賴片段分離(Methylation-dependent fragment separation; MDFS)
•基因體甲基化測試(Genomic methylation testing)
總結
分子生物技術的應用需要克服一些特定的限制,甲基化分析的
方法必須具備高靈敏度,因為體液為最普遍且容易獲得的分析材
料,其中只含有微量的腫瘤DNA,此分析方法也必須有足夠的專
一性,確保能夠區分腫瘤細胞與正常細胞的甲基化模式。
有許多方法可以分析甲基化模式,但任何方法都不是萬能的,
在選擇適合的方法時,須考慮所欲分析的生物材料的種類、數量及
品質以及所需的實驗室與儀器設備來選擇正確的方法,將有助於降
低汙染的風險和確保可重複的結果。
第二章 微衛星標記分析與應用
– 前言
• 散布重複DNA
• 串聯重複DNA
微衛星標記(Microsatellite markers)
散布重複(Interspersed repetitive DNA), 45%,
Alu repeat
串聯重複(Tandemly repetitive DNA), 10%
Satellites, minisatellites, microsatellites
微衛星,亦稱為短串聯重複(short tandem repeats, STRs; simple sequence repeats, SSRs),其是由 1∼5 個核酸為重複單位的重複性序列,例如雙核酸(dinucleotide)之 CA 重複序列:(CA)n,n 即是代表其重複次數(repeat number), 在 5∼100 之間。
微衛星的基因變異 (Genetic alterations)
• 微衛星不穩定性(Microsatellite instability; MSI)
• Mismatch repair (MMR) system (ex. MLH1, MSH1, MSH2, MSH3, MSH6, PMS2 …etc.)
• 微衛星失異合性(Loss of heterozygosity; LOH)
• Oncogenic mutations (p53, Rb, APC and DCC…etc.)
微衛星的基因變異 (Genetic alterations)
• 微衛星不穩定性(Microsatellite instability; MSI)
• Mismatch repair (MMR) system (ex. MLH1, MSH1, MSH2, MSH3, MSH6, PMS2 …etc.)
• 微衛星失異合性(Loss of heterozygosity; LOH)
• Oncogenic mutations (p53, Rb, APC and DCC…etc.)
微衛星標記分析
目前普遍使用的方法為「自動片段分析技術」
(automatic fragment analysis),乃利用不
同顏色的螢光物質標記引子進行聚合連鎖反應,並
配合自動核酸片段分析儀進行電泳與分析,大大提
升了微衛星標記分析的速度、方便性及正確性。
微衛星標記在生物醫學上的用途
• 人類身分及親源鑑定
• 疾病相關基因的搜尋
• 腫瘤診斷與治療上的應用
總結
因為微衛星多形性的特性且數量極多,加上分布於整個基因體,而分析方法的自動化、高產量與高準確性,讓微衛星標記分析已廣泛應用於各領域,包括:各種生物體演化與親緣關係研究、臨床檢驗醫學、法醫學、臨床及基礎醫學研究。特別在腫瘤醫學上可應用於多種癌症的診斷、預後、治療與追蹤,包括:大腸直腸癌、肝癌、膀胱癌、乳癌、肺癌、胃癌、子宮頸癌、卵巢癌及頭頸部鱗狀細胞癌等;主要的分析依據是觀察是否產生微衛星不穩定性或失異合性等現象;而進行分析的檢體可以是尿液、痰液、血液及手術移除的組織等。在後基因體時代中,可以預期利用微衛星分析將有助於疾病相關基因的快速定位與發現。
• 第三章 單一核苷酸多型性檢測技術之原理
與應用
Human Genome Project
• An international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000-25,000 genes of the human genome from both a physical and functional standpoint.
• Since Oct. 1990 (initiated by DOE & NIH).
• A working draft announced in 2000.
• Completed in 2003.
單一核苷酸多型性( SINGLE NUCLEOTIDE POLYMORPHISM ; SNP)
– 99.9% identical gene sequence between non-relative individuals
– 基因突變是指 DNA 分子的核苷酸序列發生變異。
– 基因突變可能源自天然輻射或是 DNA 複製錯誤而產生的自發性突變(spontaneous mutation),抑或由致突變化學物質(chemical mutagen)誘導所產生。
– DNA 變異的種類很多,包括單一或多個核苷酸的變異、插入(insertion)或缺失( d e l e t i o n ) 到染色體的重組(rearrangement)、轉位(translocation)、重複(duplication)或缺失(deletion)。
– 這些基因突變除了會改變原有之基因型外,有些突變還可能會影響生物體的表現型,甚至引起遺傳性疾病。
SNP 檢測技術之基礎原理
– 檢測與疾病或特殊表現型有關的基因突變以及 SNP 是分子診斷學中一項重要的功能。雖然 DNA 序列分析(DNA sequencing)是檢測基因突變最可靠的方法,但是無法一次檢測大量的 SNP。
– 目前已有許多不需解讀 DNA 序列就可偵測SNP 的技術,最常用之檢測技術基礎原理主要為雜交反應(hybridization)與聚合連鎖反應(polymerase chain reaction)。
• 雜交反應
• 聚合連鎖反應
– SNP 檢測技術
• 以雜交反應為檢測基礎的技術
– 單股結構多型性(single-strand conformation polymorphisms; SSCP)單股結構多型性(SSCP)技術就是根據單股 DNA 分子構形取決於其核苷酸序列的理論基礎,在特定之 DNA 變性溫度等條件下,不同序列構形在電泳膠片中的移動速率也會因此產生變化,用以偵測待測檢體 DNA 片段的 SNP。
變性梯度膠體電泳法 (denaturing gradient gel electrophoresis; DGGE)
Frequently used denaturant: urea and formamide.
Parallel水平式 Perpendicular 垂直式
聚合酶連鎖反應—對偶基因特異性雜交反應(PCR-allele specific oligonucleotide
hybridization; PCR-ASO) 首先使用 PCR 大量複製雙股 DNA 待測檢體,然後將待測檢體之PCR 產物固定在硝化纖維膜上。再利用含有高濃度鹽類的鹼性變性溶液浸泡硝化纖維膜,使膜上的雙股 DNA 分子變性成單股DNA。之後使用已標定並和正常序列或是 SNP 突變序列互補的探針分別與膜上的待測 DNA 分子進行雜交反應。反應結束後,就可依照雜交反應結果判斷待測檢體DNA 是否帶有 SNP 變異。
Binding of “S” ASO probe to “S” DNA
Binding of “S” ASO probe to “A” DNA
溶解曲線分析法 (melting curve analysis; MCA)
溶解曲線分析利用即時 PCR(Real-time PCR)技術,反應時加入可和雙股 DNA 作特異性結合的溴化乙錠(EtBr)或 SYBRgreen I 等染劑,並在 PCR 反應過程中定速地逐漸提高溫度(大約 3°C/秒)。在反應剛開始時因為溫度較低,待測檢體的 PCR 產物都會和染劑結合到 PCR 產物,使訊號較強。當 PCR 反應溫度隨著複製循環數逐漸升高過程中,雙股 DNA 分子便逐漸變性為單股 DNA,因此 PCR 產物會和染劑分開而使訊號強度慢慢減弱。所以可根據 PCR 複製過程中結合到雙股 DNA 分子訊號強度的減弱情況,判斷 DNA 待測檢體是否有 SNP。
Genotyping of two adjacent SNPs (HbS and HbC) within a 110-bp β-globin fragment with use of high-resolution amplicon
melting curve analysis.
以 DNA 合成反應為檢測基礎的技術 – 序列特異性 PCR(sequence-specific PCR;SS-PCR)
以 DNA 合成反應為檢測基礎的技術 – 雙去氧核糖核酸指紋分析(dideoxy DNA fingerprinting)
• 以酵素切割反應為檢測基礎的技術
– 聚合酶連鎖反應—限制片段長度多型性分析(polymerase chain reactionrestriction fragment length polymorphisms;PCR-RFLP)
– 單股 DNA 特異性核酸分析(Heteroduplex analysis with single-strand specific nucleases)
微陣列技術:可同時檢測多種 SNP 的技術
DNA microarrays 是 1990 年代所發展出的技術,將正常 DNA 序列與已知的 SNP 序列結合在矽晶片或玻璃等材質上,每一平方公分約可結合一百萬種 DNA 序列,然後和已標定之待測檢體(基因體DNA 片段)進行雜交反應。因為可將已知的 DNA 變異序列都固定在晶片上,所以就可從單次雜交反應結果的同時,得知某一個體所帶有的 SNP 組合資料。目前偵測 P53 抑癌基因突變之微陣列技術的敏感度與特異性,已可媲美 DNA 序列分析。微陣列技術的優點是可同時一次檢測單一基因之多種基因突變位,也可同時檢測多種基因的突變。
SNP microarray
– SNP 在醫學領域的應用
• 人類白血球抗原分型與移植醫學
HLA 基因座中的遺傳密碼是負責建立免疫系統自我辨
識的胜肽鏈,所以器官捐贈者與授受者的免疫系統
DNA 序列必須有相似性或是相容性才能進行器官移植
手術。
藥物基因體學(Pharmacogenomics)與個人化醫療(Personalized medicine)
• 檢測疾病相關基因
除了器官移植與藥物反應之外,已知某些遺傳性疾病與 SNP 有密不可分之相關性。鐮刀型貧血(sickle cell anemia)是因為負責製造血紅素之 β2 球蛋白基因的第6 組密碼產生點突變,而製造異常血紅素Hb-S。Hb-S 分子與正常血紅素 Hb-A 結構不同,使其與氧氣結合之親和力下降,導致紅血球呈鐮刀狀外形與組織損傷 。目前臨床上已可使用 PCR-RFLP 檢測遺傳性鐮刀型貧血等疾病的 SNP。對疾病與致病基因的了解與認識更透徹,則更能正確地診斷、預測與治療潛在疾病,增進人類福祉。