DNA Technology & Biotechnology
Transcript of DNA Technology & Biotechnology
![Page 1: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/1.jpg)
DNA Technology & Biotechnology
DNA Technology develops applications based on basic understanding of the components of molecular genetics
- DNA replication- Transcription- Translation- Control systems regulating all these
processes
![Page 2: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/2.jpg)
Gene cloning aims at making a large number of copies of a particular piece of DNA:- within a living organism (in vivo)- in a test tube (in vitro)
![Page 3: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/3.jpg)
• Some basic tools of DNA technology
- Genomic DNA- Vectors as shuttles for genes between
organisms: Plasmids or viruses- Enzymes to manipulate DNA: ligase,
DNA polymerase, & Restriction enzymes- Gel electrophoresis
![Page 4: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/4.jpg)
Plasmids• Extrachromosomal circular DNA • Found in most bacteria• Multiply independently of bacterial chromosome• Carry useful but non-essential genes, extend the biochemical capabilities of bacteria (antibiotic resistance genes)
![Page 5: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/5.jpg)
Biotechnology uses DNA technology to produce useful products
Vectors: - Cloning vectors- no promoter- Expression vectors- with promoter to produce the desired proteins
Living cells:- Bacteria- Yeast- Several eukaryotic cells
![Page 6: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/6.jpg)
Restriction enzymes http://highered.mcgraw-
hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Gel electrophoresishttp://www.lewport.wnyric.org/jwanamaker/a
nimations/Chrom%26Elpho.html
![Page 7: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/7.jpg)
![Page 8: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/8.jpg)
![Page 9: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/9.jpg)
• Restriction Fragment length Polymorphisms (RFLPs)
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
![Page 10: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/10.jpg)
DNA Fingerprinting
• Isolation of DNA• Cutting, sizing, and sorting DNA. Special
enzymes called restriction enzymes• Producing a DNA profile of fragments that
appear as bands (using many alternativetechniques..)
![Page 11: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/11.jpg)
Practical Applications of DNAFingerprinting
• Paternity and Maternity• Criminal Identification
and Forensics• Personal Identification
![Page 12: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/12.jpg)
Practical Applications of DNAFingerprinting
“Forensic Biotechnology Whodunit?” by Jenny Shaw, Vanessa Petty, Theresa Brown, and Sarah Mathiason
![Page 13: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/13.jpg)
Practical Applications of DNAFingerprinting
![Page 14: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/14.jpg)
DNA Technology & Biotechnology
Background Information
• Definitions of DNA technology, gene cloning (in vivo & in vitro), genetic engineering & biotechnology
• Basic tools of biotechnology: DNA isolation, plasmids, restriction enzymes, DNA gel electrophoresis, transformation
• Gene cloning versus protein expression vectors
Basic Lab Skills
• Plasmid components & map• Use of micropipettor• Setting-up restriction enzyme
digests of a plasmid• Pouring agarose gels-• Preparing digestion for gel
loading• Use of molecular weight
markers and running DNA electrophoresis gels
• Analysis of results
![Page 15: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/15.jpg)
Background InformationPlasmids
• Source, composition & architecture• Genes found on plasmids• Functional DNA sequences of
plasmids: o origin of replicationo selectable marker- antibiotic
resistance gene of interesto Gene(s) to be clonedo Control elements for gene
expression
• Plasmid map
Restriction Enzymes• Where are they naturally found?• Biological function• Enzymatic activity- catalysis of
sequence-specific cutting of DNA
• Staggered cutting-sticky ends• Blunt cutting-no sticky ends
Plasmid Maps with Restriction Enzyme sites• Size of plasmid• Position of restriction enzymes sites• Size of fragments when cut and genes they carry
![Page 16: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/16.jpg)
DNA Gel ElectrophoresisBackground Information
• Agarose gels composition• Ethidium bromide• Negative charge of DNA • Direction of migration during
electrophoresis• Effect of size of linear DNA on its
migration• Molecular weight size marker• Gel- loading Dye (glycerol and
tracking dye) • Migration of non-linear DNA
fragments: super-coiled and relaxed circles
• Size determination of linear fragments
Basic Lab Skills
• Plasmid components & map• Use of micropipettor• Setting-up restriction enzyme
digests of a plasmid• Pouring agarose gels• Running gel electrophoresis• Results and Analysis
![Page 17: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/17.jpg)
DNA Gel Electrophoresis Results & Analysis
Background Information & Basic Lab Skills
• 1 kb plus ladder- fragment sizes
• Migration of super-coiled and relaxed circles of plasmids
• Comparison of pattern of fragments for uncut and cut plasmid (BamHI-HindIII)
Questions
x
x
1 kb plus Ladder
![Page 18: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/18.jpg)
Transformation Background
• Host cell• Cloning only vectors and expression vectors
Origin of replicationPromoters-
genes always expressed- promoter open for RNA polymerase bindinggenes with controlled expression- RNA polymerase binding under certain conditions
• Gene expression in bacteriaOperons Transcription control elements
![Page 19: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/19.jpg)
Transformation LabBackground Information
• Host cell & plasmid (rpARA)• Expression of ampr gene-
selectable marker• Expression of the RFP (tomato
gene)• Natural competence- ability to take
up naked DNA from surrounding• Induced competence-CaCl2
treatment• Heat shock• Recovery• Plating & growing of bacterial cells
Basic Lab Skills
• Understanding of experimental set-up
• Follow written instruction of the Transformation protocol
• Answer Questions• Results and analysis
![Page 20: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/20.jpg)
![Page 21: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/21.jpg)
The Arabinose Operon
RFP (tomato)
![Page 22: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/22.jpg)
PCRBackground Information
• Genomic DNA and cell lysis• Components of lysis mixture• Review DNA replication in vivo • DNA replication in vitro• Compare in vivo & in vitro DNA
synthesis• PCR
• Steps • Reaction mixture components
• Human genomic DNA to be amplified
Basic Lab Skills
• Genomic DNA preparation• Setting-up a PCR reaction
• Analysis using DNA Gel Electrophoresis
![Page 23: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/23.jpg)
Compare and contrast DNA Replication in vivo
PCR-DNA Amplification in vitro
![Page 24: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/24.jpg)
DNA Replication
• InitiationStrand Separation by helicasePrimer synthesis by primase
• ElongationExtension and polymerization of nucleotides by DNA polymerase using dNTPs: dATP, dTTP, dCTP, and dGTP
• TerminationLeading strand- End of template strandLagging strand- Replace RNA primer with DNA strand & fill-in gaps with DNA ligase
![Page 25: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/25.jpg)
Comparison of In vivo and In vitro reactions
In vivo In vitro
Strand separation –
Priming –
Elongation –
![Page 26: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/26.jpg)
Preparation of genomic DNA
• Lyse or break cells by disrupting cell membranes using a detergent
• Digest with a protease to release naked DNA
• Separate DNA from digested proteins
![Page 27: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/27.jpg)
PCR: polymerase Chain ReactionDNA amplification in a test tube
http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
![Page 28: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/28.jpg)
PCR animation Dolan learning Center• http://www.dnalc.org/
• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Polymerase Chain Reaction (PCR)http://www.biology.arizona.edu/
![Page 29: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/29.jpg)
• cDNAhttp://highered.mcgraw-
hill.com/sites/0072437316/student_view0/chapter16/animations.html#
• Plasmid cloninghttp://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html
• Steps of gene cloning http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Plasmid Cloning
![Page 30: DNA Technology & Biotechnology](https://reader030.fdocuments.net/reader030/viewer/2022040404/624855cacf5bd30f8b13c3c5/html5/thumbnails/30.jpg)
Recombinant DNA Technology
• http://present.smith.udel.edu/biotech/rDNA.html
Plasmid cloning and transformation• http://www.sumanasinc.com/webcontent/a
nisamples/molecularbiology/plasmidcloning_fla.html