DNA replication and genetic engineering

傅淑玲老師

陽明大學傳醫所

電話:28267177email: slfu@ym.edu.tw

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