Recombinant DNA
Technology
and
Genomics
The impact of recombinant DNA technology
Recombinant DNA technology
•A set of methods used to locate, analyze, alter, study
and recombine DNA sequence
•probe the structure and function of genes
•Create commercial products, diagnose and treat disease
Cutting and joining DNA fragments
Restriction enzymes: 限制酶
Cohesive ends
Viewing DNA fragments
Under UV light
Cloning genes
Cloning vector: 選殖載體
2.
1.
3.
重組後的質體
質體種類
YAC: yeast artificial chromosome
Contain a yeast origin of replication
Creating a genomic library:
finding genes
Genomic library:
A collection of DNA
Sequences found in an
Organism’
s genome
cDNA library
All transcribed
genes
Find the gene of interest:
Screen library with a probe
DNA amplification: Polymerase chain reaction
A hot spring in Yellowstone
National Park in US
Thermus aquaticus
Analyzing DNA sequence
In situ dybridization
Green probe: X chromosome
Red probe: Y chromosome
mRNA at both ends
DNA footprint
Determine which DNA
Sequences are bound
by the protein
Site-directed mutagenesis
基因轉殖技術的應用
•人類疾病的動物模型
•以完整的動物模式研究基因的功能
•疾病基因的互補性測試
•藥物與生技產品的應用性
–例如：藥物篩選或生產人類的抗體
基因轉殖鼠的實驗方法
•Injection of DNA into pronucleus of
fertilized mouse egg
•Transfection of embryonic stem (ES)
cells with DNA constructs and injection
of ES cells into blastocyst embryo
•Viral infection of fertilized egg or
blastocyst embryo
•Somatic cell nuclear transfer (SCNT)mediated gene transfer
Transgenic animals
Randomly integrated
To chromosome by
Nonhomologous recombination
體細胞與複製羊
•Somatic cell nuclear transfer
Dolly the sheep
Knockout mice
Applications of Recombinant DNA Technology
•Pharmaceuticals
•Specialized bacteria
•Agricultural products
•Oligonucleotide drugs
•Genetic testing
•Gene therapy
•Gene mapping
•DNA fingerprinting
Oligonucleotide drugs: short DNA or RNA
Gene therapy: treat genetic disease, cancer, heart disease
RFLPs:
Restriction fragment
length polymorphisms
Coinheritance of the gene
and an RFLP
Huntington-disease gene was mapped to chromosome 8
By RFLP
Banding patterns revealed by variation in
microsatellite sequences
DNA
fingerprinting
The power of genomics
Leprosy, a disease caused by
Mycobacterium lepreae
Genomics:
DNA sequencing
Map-based approach
Whole-genome shotgun
sequencing
Approach 1
Approach 2
The entire genome: Haemophilas influenzae
The first free-living organism
to be sequenced
請閱讀 p. 565 的文章，將其翻譯成中文，並寫下
500~1000 的感想。
期末考週為最後繳交期限。
The New Genetics
The Human Genome Project:
Book of Life or Pandora’
s Box?
Single-nucleotide polymorphisms
Single-base-pair differences in DNA sequence
between individual members of a species
Bioinformatics
Functional genomics
Predicting function from sequence:
Homology search
A1 and A2: paralogs
B1 and B2: paralogs
A1 and B1: orthologs
A2 and B2: orthologs
Gene expression and Microarrays
Comparative genomics
•Allowing inferences about how genes function
and genomes evolve
•provide important information about evolutionary
Relationship among organisms
•and about factors that influence the speed and
Direction of evolution
Two circular chromosomes:
Vibrio cholerae
One large linear chromosome
And 21 smaller chromosomes:
Borrelia burgdorferi
Gene number and complexity
Repetitive sequences and noncoding sequences
Protein domains and protein diversity
•Assembled domains into different combinations
•Homologs in distantly related species
•
Mouse and human (99% in common)
•Alternative splicing
•Gene density
•A variety of transposable elements