Lecture #9Date _____ n Chapter 20~ DNA Technology & Genomics.

Lecture #9Lecture #9 Date _____Date _____

Chapter 20~Chapter 20~DNA DNA

Technology Technology & & GenomicsGenomics

O.J. Simpson capital murder case,1/95-9/95 Odds of blood in Ford Bronco not being R. Goldman’s:

6.5 billion to 1 Odds of blood on socks in bedroom not being N. Brown-Simpson’s:

8.5 billion to 1 Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:

21.5 billion to 1 Number of people on planet earth:

6.1 billion Odds of being struck by lightning in the U.S.:

2.8 million to 1 Odds of winning the Illinois Big Game lottery:

76 million to 1 Odds of getting killed driving to the gas station to buy a lottery ticket

4.5 million to 1 Odds of seeing 3 albino deer at the same time:

85 million to 1 Odds of having quintuplets:

85 million to 1 Odds of being struck by a meteorite:

10 trillion to 1

Recombinant DNARecombinant DNA

Def:Def: DNA in which genes DNA in which genes from 2 different sources from 2 different sources are linkedare linked

Genetic engineering:Genetic engineering: direct manipulation of direct manipulation of genes for practical genes for practical purposespurposes

Biotechnology:Biotechnology: manipulation of organisms manipulation of organisms or their components to or their components to perform practical tasks or perform practical tasks or provide useful productsprovide useful products

Bacterial plasmids in gene cloningBacterial plasmids in gene cloning

DNA CloningDNA Cloning Restriction enzymes (endonucleasesRestriction enzymes (endonucleases):): in in

nature, these enzymes protect bacteria from nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); intruding DNA; they cut up the DNA (restriction); very specificvery specific

Restriction siteRestriction site::recognition sequence for a particular restriction recognition sequence for a particular restriction enzymeenzyme

Restriction fragmentsRestriction fragments::segments of DNA cut by restriction enzymes in a segments of DNA cut by restriction enzymes in a reproducable wayreproducable way

Sticky endSticky end::short extensions of restriction fragmentsshort extensions of restriction fragments

DNA ligaseDNA ligase: : enzyme that can join the sticky ends of DNA enzyme that can join the sticky ends of DNA fragmentsfragments

Cloning vectorCloning vector: : DNA molecule that can carry foreign DNA into a DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial cell and replicate there (usually bacterial plasmids)plasmids)

Restriction EnzymesRestriction Enzymes

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Steps for eukaryotic gene cloningSteps for eukaryotic gene cloning

Isolation of cloning vector Isolation of cloning vector (bacterial plasmid) & gene-(bacterial plasmid) & gene-source DNA (gene of interest)source DNA (gene of interest)

Insertion of gene-source DNA Insertion of gene-source DNA into the cloning vector using the into the cloning vector using the same restriction enzyme; bind the same restriction enzyme; bind the fragmented DNA with DNA ligasefragmented DNA with DNA ligase

Introduction of cloning vector into Introduction of cloning vector into cells (transformation by bacterial cells (transformation by bacterial cells)cells)

Cloning of cells (and foreign Cloning of cells (and foreign genes)genes)

Identification of cell clones Identification of cell clones carrying the gene of interestcarrying the gene of interest

CloningCloning

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DNA Analysis & GenomicsDNA Analysis & Genomics

PCR (polymerase chain PCR (polymerase chain reaction)reaction)

Gel electrophoresisGel electrophoresis Restriction fragment Restriction fragment

analysis (RFLPs)analysis (RFLPs) Southern blottingSouthern blotting DNA sequencingDNA sequencing Human genomeHuman genome projectproject

Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)

Amplification of any Amplification of any piece of DNA without piece of DNA without cells (in vitro)cells (in vitro)

Materials: heat, DNA Materials: heat, DNA polymerase, polymerase, nucleotides, single-nucleotides, single-stranded DNA primersstranded DNA primers

Applications: fossils, Applications: fossils, forensics, prenatal forensics, prenatal diagnosis, etc.diagnosis, etc.

DNA AnalysisDNA Analysis

Gel electrophoresisGel electrophoresis: separates nucleic acids or proteins on the : separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the basis of size or electrical charge creating DNA bands of the same lengthsame length

Restriction fragment analysisRestriction fragment analysis

Restriction fragment length polymorphisms (RFLPs)Restriction fragment length polymorphisms (RFLPs) Southern blottingSouthern blotting: process that reveals sequences and : process that reveals sequences and

the RFLPs in a DNA sequencethe RFLPs in a DNA sequence DNA FingerprintingDNA Fingerprinting

DNA SequencingDNA Sequencing

Determination of Determination of nucleotide sequences nucleotide sequences (Sanger method, (Sanger method, sequencing machine)sequencing machine)

Genomics: the study of Genomics: the study of genomes based ongenomes based onDNA sequencesDNA sequences

Human Genome Human Genome ProjectProject

Practical DNA Technology UsesPractical DNA Technology Uses

Diagnosis of diseaseDiagnosis of disease Human gene therapyHuman gene therapy Pharmaceutical products Pharmaceutical products

(vaccines)(vaccines) ForensicsForensics Animal husbandry Animal husbandry

(transgenic organisms)(transgenic organisms) Genetic engineering in plantsGenetic engineering in plants Ethical concerns?Ethical concerns?