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독뉑곂ꛑ깶 뚧ꧺꑪ뻇뛇싥꧒ 륱룜:28267177 email: [email protected]
Transcript of 독뉑곂ꛑ깶 뚧ꧺꑪ뻇뛇싥꧒ 륱룜:28267177 email: [email protected]
參考文獻:
1. Nelson, D. & Cox, M. (2000) “Lehninger Principles of biochemistry”, 3rd editionChapter 10, 25 & 29
2. Alberts, B., Bray, D., Johnson, A., Lewis, J., Raff, M.,Roberts, K. & Walter, P. (1997)“Essential Cell Biology”, Chapter 6 &10
DNA stores and transmits genetic information
H(RNA)(DNA)
Structure of nucleotides
Chemical bonding in DNA
Hydrogen-bonding patterns in the base pairs
Phosphodiester-bonding linkage
DNA structure
- The structure of DNA is a double-stranded antiparallel helix- DNA molecules have distinctive base composition - Nuleic acids hybridize by base pairing
DNA replication (DNA synthesis)
DNA replication is semi-conservative DNA synthesis of eukaryotic cells is bi-directional and starts at multiplereplication origin
DNA replication
Asymmetry of strand synthesis during DNA replication- DNA synthesis is semidiscontinuous
- DNA synthesis direction: 5' to 3'
DNA replication
- DNA polymerases: the enzymes that make DNA - DNA polymerization by DNA polymerase requires templates,
primers and deoxynucleotides
(dNMP)n + dNTP (dNMP)n + PPi+1
Major enzymes participates in DNA replication
How DNA molecules are analyzed ?
- Restriction endonucleases cut DNA molecules at specific sites
- Gel electrophoresis separates DNA fragments of different sizes
- The nucleotide sequence of DNA can be determined
- Chemical synthesis of DNA has been automated
Chemical synthesis of DNA has been automated
Restriction endonucleases cut DNAmolecules at specific sites
Sticky ends vs. blunt ends
Gel electrophoresis separates DNA fragments of different sizes
Ethedium bromide staining
Autoradiography
The nucleotide sequence of DNA can be determined
DNA sequencing by the Sanger method
Strategy for automating DNA sequencing reactions
Genetic engineering ( Recombinant DNA technology )
DNA cloning
- Isolating a gene from a cellular genome
Cloning vectors:allow amplification of inserted DNA segments in cells
- Plasmid- Bacteriophage- Bacterial artificial chromosome (BAC)
Restriction enzymedigestion
Ligation
Transformation
Selection and amplification ofrecombinant DNA
- Creating recombinant DNA
Creating recombinant DNA
Key enzymes: restriction enzymes and DNA ligase
Bacteriophage as a cloning vector
Cloning vectorpBR322
Specific DNA sequence can be amplified by polymerase chain reaction (PCR)
Principle: In vitro DNA synthesis usingthermostable DNA polymerase
denaturationDNA synthesis
annealing
denaturationannealing
DNA synthesis
The PCR cycle
Use PCR to detect the presence of a viralgenome in a sample of blood
Isolating a gene from a cellular genome
Cloning a gene often requires a DNA library
- genomic library- cDNA library
cDNA synthesis requires m RNAand reverse transcriptase
- reverse transcriptase:a RNA-directed DNA polymerase
DNA library
Synthesis of complementary DNA (cDNA)
DNA libraries
Use of PCR to obtain a genomic or cDNA clone
Identifying a clone with a particularDNA segment using hybridization
Hybridization with probe is a sequence-based process to detect a particular cDNA fragment
DNA hybridizationColony hybridization
DNA microarrays provide compact librariesfor studying gene expression
Analysis of differentially expressed genes
Cloned genes can be expressed
Production of large amounts of a protein from a protein-coding sequencecloned into an expression vectors and introduced into cells
Designing a probe to detect the gene for a protein of known amino acid sequence
Mutant organisms best reveal the function of a gene
Three common ways to reveal the function of a specific gene intransgenic organisms