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

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)

• 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

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)

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

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

• Restriction Fragment length Polymorphisms (RFLPs)

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

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..)

Practical Applications of DNAFingerprinting

• Paternity and Maternity• Criminal Identification

and Forensics• Personal Identification

Practical Applications of DNAFingerprinting

“Forensic Biotechnology Whodunit?” by Jenny Shaw, Vanessa Petty, Theresa Brown, and Sarah Mathiason

Practical Applications of DNAFingerprinting

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

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

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

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

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

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

The Arabinose Operon

RFP (tomato)

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

Compare and contrast DNA Replication in vivo

PCR-DNA Amplification in vitro

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

Comparison of In vivo and In vitro reactions

In vivo In vitro 

Strand separation –

Priming –

Elongation –

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

PCR: polymerase Chain ReactionDNA amplification in a test tube

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

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/

• 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

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