chapter 20~dna technology & genomics. who am i? recombinant dna n def: dna in which genes from 2...

Chapter 20~Chapter 20~DNA Technology & DNA Technology & GenomicsGenomics

Who am I? Who am I?

Recombinant DNARecombinant DNA

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

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

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

Bacterial plasmids in gene cloningBacterial plasmids in gene cloning

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

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

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

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

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

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

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

Restriction EnzymesRestriction Enzymes

Steps for eukaryotic gene cloningSteps for eukaryotic gene cloning Isolation of cloning vector (bacterial plasmid) Isolation of cloning vector (bacterial plasmid)

& gene-source DNA (gene of interest)& gene-source DNA (gene of interest) Insertion of gene-source DNA into the cloning Insertion of gene-source DNA into the cloning

vector using the same restriction enzyme; bind vector using the same restriction enzyme; bind the fragmented DNA with DNA ligasethe fragmented DNA with DNA ligase

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

Cloning of cells (and foreign genes)Cloning of cells (and foreign genes) Identification of cell clones carrying the gene Identification of cell clones carrying the gene

of interestof interest

CloningCloning

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 genome projectHuman genome project

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 : separates nucleic

acids or proteins on the basis of size or acids or proteins on the basis of size or electrical charge creating DNA bands of electrical charge creating DNA bands of the same lengththe same length

Restriction fragment analysisRestriction fragment analysis

Restriction fragment length polymorphisms Restriction fragment length polymorphisms (RFLPs)(RFLPs)

Southern blottingSouthern blotting: process that reveals : process that reveals sequences and the RFLPs in a DNA sequencesequences and the 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

Sanger MethodSanger Method

http://www.dnalc.org/view/15479-Sanger-method-of-DNA-sequencing-3D-animation-with-narration.html

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 Genetic engineering in

plantsplants Ethical concerns?Ethical concerns?