DNA Technology n Now it gets real….. O.J. Simpson capital murder case,1/95-9/95 Odds of blood on socks in bedroom not being N. Brown-Simpson’s: 8.5 billion.

DNA TechnologyDNA Technology

Now it gets real….Now it gets real….

O.J. Simpson capital murder case,1/95-9/95 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 PowerBall 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

Recombinant DNARecombinant DNA Definition: Definition: DNA in which DNA in which

genes from 2 different genes from 2 different sources are linkedsources are 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 nature, these enzymes protect in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA bacteria from intruding DNA; they cut up the DNA (restriction); very specific (restriction); very specific link to animationlink to animation

Restriction siteRestriction site:: recognition recognition sequence for a particular restriction enzymesequence for a particular restriction enzyme

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

Sticky endSticky end:: short extensions of short extensions of restriction fragments resulting from the restriction fragments resulting from the “staggered cut”“staggered cut”

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

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

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 same restriction enzyme; bind the fragmented DNA with DNA the fragmented DNA with DNA ligaseligase

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

Campbell LinkCampbell Link

DNA Analysis & GenomicsDNA Analysis & Genomics

PCR (polymerase chain PCR (polymerase chain reaction) reaction) link to PCR animationlink to PCR animation

Gel electrophoresis Gel electrophoresis electrophoresis animation linkelectrophoresis animation link

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 primers stranded DNA primers PCR animation linkPCR animation link

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 electrophoresis animation linkelectrophoresis animation link

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, Sanger link 1Sanger link 1 Sanger link 2Sanger link 2

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?