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Berg • Tymoczko • Stryer Biochemistry Sixth Edition Chapter 5: Exploring Genes and Genomes Berg • Tymoczko • Stryer Biochemistry Sixth Edition Chapter 5: Exploring Genes and Genomes Copyright © 2007 by W. H. Freeman and Company

5. 1 The exploration of genes relies on key tools Restriction enzyme analysis Blotting 5. 1 The exploration of genes relies on key tools Restriction enzyme analysis Blotting techniques DNA sequencing Solid-phase synthesis Polymerase chain reaction

Restriction enzyme split DNA into specific fragments Restriction and modification system Palindrome Restriction enzyme split DNA into specific fragments Restriction and modification system Palindrome

Sac. II Sac. II

Restriction fragments can be separated by gel electrophoresis and visualized Agarose or polyacrylamide gel Restriction fragments can be separated by gel electrophoresis and visualized Agarose or polyacrylamide gel One nucleotide difference Ethidium bromide, autoradiography, silver staining Southern blotting and Northern blotting DNA probe

DNA can be sequenced by controlled termination of replication (Sanger dideoxy method) Controlled interruption DNA can be sequenced by controlled termination of replication (Sanger dideoxy method) Controlled interruption of enzymatic replication Add 2’, 3’ dideoxy analog of one of the nucleotides Fluorescence detection

DNA probes and genes can be synthesized by automated solid-phase method Activated monomer: deoxyribonucleoside DNA probes and genes can be synthesized by automated solid-phase method Activated monomer: deoxyribonucleoside 3’-phosphoramidite Phosphite triester Phophotriester Blocking group: dimethoxytrityl (DMT) and b-cyanoethyl (b. CE)

Selected DNA sequences can be greatly amplified by the polymerase chain reaction Primers, d. Selected DNA sequences can be greatly amplified by the polymerase chain reaction Primers, d. NTP, heat-stable DNA polymerase Strand separation Hybidization of primers DNA synthesis

PCR is a powerful technique in medical diagnostics, forensics, and molecular evolution PCR is a powerful technique in medical diagnostics, forensics, and molecular evolution

5. 2 Recombinant DNA technology has revolutionized all aspects of biology Restriction enzymes and 5. 2 Recombinant DNA technology has revolutionized all aspects of biology Restriction enzymes and DNA ligase are key tools in forming recombinant DNA molecule Cohesive or sticky ends Blunt ends DNA ligase DNA linker

Plasmids and lambda phage are choice vectors for DNA cloning in bacteria p. BR Plasmids and lambda phage are choice vectors for DNA cloning in bacteria p. BR 322 plasmid, p. UC 18 plasmid Lambda phage: lytic or lysogenic pathway In vitro packaging cosmid M 13 phage: (+) strand, RF

Bacterial and yeast artificial chromosomes BAC and YAC Bacterial and yeast artificial chromosomes BAC and YAC

Specific genes can be cloned from digests of genomic DNA Genomic library Screening a Specific genes can be cloned from digests of genomic DNA Genomic library Screening a library with a specific probe

Proteins with new functions can be created through directed changes in DNA Deletions, substitutions, Proteins with new functions can be created through directed changes in DNA Deletions, substitutions, insertions Oligonucleotide-directed mutagenesis Cassette mutagenesis Designer genes

5. 3 Complete genomes have been sequenced analyzed The genomes of organisms ranging from 5. 3 Complete genomes have been sequenced analyzed The genomes of organisms ranging from bacteria to multicellular eukaryotes have been sequenced

Haemophilus influenzae 뇌수막염 Haemophilus influenzae 뇌수막염

The sequencing of the human genome has been finished 25, 000 genes A lot The sequencing of the human genome has been finished 25, 000 genes A lot of pseudogenes Mobile genetic elements: Short interspersed elements (SINES) – Alu sequence Long interspersed elements (LINES)

Comparative genetics has become a powerful research tools Comparative genetics has become a powerful research tools

Gene expression levels can be comprehensively examined DNA microarray or gene chip Gene expression levels can be comprehensively examined DNA microarray or gene chip

5. 4 Eukaryotic cells can be manipulated with considerable precision Complementary DNA prepared from 5. 4 Eukaryotic cells can be manipulated with considerable precision Complementary DNA prepared from m. RNA can be expressed in host cells c. DNA library Reverse transcriptase, terminal transferase Expression vectors

New genes inserted into eukaryotic cells can be efficiently expressed transfection: Ca. PO 4, New genes inserted into eukaryotic cells can be efficiently expressed transfection: Ca. PO 4, liposome microinjection viral vectors Vaccinia virus Baculovirus

Transgenic animals harbor and express genes that were introduced into their germ lines Metallothionein Transgenic animals harbor and express genes that were introduced into their germ lines Metallothionein induced by heavy metal ions Microinjected into the male pronucleus of a fertilized mouse egg Transgenic mice

Production of transgenic mice Production of transgenic mice

Gene disruption provides clues to gene function Gene disruption (gene knockout) Homologous rcombination Ex) Gene disruption provides clues to gene function Gene disruption (gene knockout) Homologous rcombination Ex) KO of myogenin gene

Gene knockout in mice Gene knockout in mice

RNA interference provides an additional tool for disrupting gene expression RNA interference si. RNA-induced RNA interference provides an additional tool for disrupting gene expression RNA interference si. RNA-induced silencing complex (RISC)

Tumor-inducing plasmids can be used to introduce new genes into plant cells Agrobacterium and Tumor-inducing plasmids can be used to introduce new genes into plant cells Agrobacterium and crown gall Ti (tumor-inducing) plasmid Electroporation of protoplast Gene gun Genetically modified organism (GMO)

Human gene therapy holds great promise for medicine Deficiency of adenosine deaminase Human gene therapy holds great promise for medicine Deficiency of adenosine deaminase