Key Terms

stubbornnessuglyBiotechnology

Dec 12, 2012 (4 years and 8 months ago)

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Introduction




Biotechnology

o

Recombinant DNA

o

Genetic Engineering



Limitations to DNA Research

o

Molecule Size

o

Gene to Chromosome Size

o

Noncoding Sequences

o

Gene to Non
-
Gene Distinctions


DNA Extraction



Membrane Dissolution



Centrifuge & Cell Fractionalization


DNA Cloning & Expression



Restriction Enzymes

& DNA Fragmentation

o

Restriction Site (4
-
8 bases)

o

Restriction Enzyme Specificity & Natural Short Sequence Repetition


Restriction Fragments

o

Sticky End Restrictors

o

DNA Ligase



DNA Cloning

o

Plasmids

o

Plasmid
Modification & Bacterial Transformation by Foreign DNA

o

Recombinant DNA & Genetic Engineered Organism

o

Cell Cycle & Gene Cloning

o

Cloning Vector

o

Using Restriction Enzymes to to Mix/Alter Plasmids

o

Plate Identification



DNA Libraries

o

Genomic



Basics



Specific Ba
cterial Plasmids



Plasmid Cone


Genomic Library



Bacteriophage



Bacterial Artificial Chromosomes (BAC)



Multiwell storage



Advantages



Best for unknown genes & unmapped genes



Regulatory & Intron sequences included



cDNA

o

Steps



Mature mRNA



Primers
+ Bases + Enz
ymes



Reverse Transcription



mRNA Degradation



DNA Polymerase + Primer



cDNA

o

Advantages



Specific protein / phenotype



Studying specific cells



Best for known gene / mapped gene



Study gene expression patterns by repeated observation of expressing cell



DNA Library

Screening

(DNA Hybridization)

o

DNA Hybridization

o

Nucleic Acid Probe (Radioactively tagged / Dyed)

o

Multiwell


Probe coated nylon membrane


Identified strand



Cloned DNA Expression

o

Expression Vector

o

Yeast vs. Bacteria Cells



RNA processing



Post
-
Translational Modification

o

DNA Insertion Techniques



Phage



Electroporation



Heat Shock



Bacteria (e.g. Agrobacterium in plants)

o

Cross Gene Expression & Evolutionary Ancestry


DNA Amplification



Polymerase Chain Reaction



Advantages

o

Skips Library Formation
& Screening Step

o

Faster

o

No cell culturing

o

No plate identification



Parts

o

Heat stable DNA Polymerase (Taq DNA Polymerase from hot spring bacteria)

o

Heat induced primed DNA molecule

o

Repeated cycles (2
n

correct sequence
, n = cycle number
)



Disadvantages

o

Errors

o

Sequence length limitation

o

Need to sequence array to identify error free fragments



Applications

o

DNA Fingerprinting

o

Species genomic analysis & sequencing

o

Prenatal diagnosis

o

Cancer genomic analysis

o

Viral genomic analaysis


DNA Sequencing & Analysis




DNA S
equencing

o

Applications of DNA Sequencing



Comparative genome analysis



Origin of phonotypical differences (epigenetics)



Phylogenetic & Evolutionary Timeline



Central Dogma Analysis



Temporal and local gene expression analysis



Gene function

o

Electrophoresis

(Separating DNA)



Agorose gel



Electrostatic potential / magnetic flow (Phosphate



Gel attraction / Density & Fragment travel rate



Resolution power



DNA Cloning + Fragmentation



DNA fragment separation



Restriction fragment length polymorphism

o

Southern Blotti
ng



Eletrophoresis + DNA Hybridization with Radioactive probe

o

Dideoxy Chain Termination Sequencing (Next
-
Generation Sequencing)

-

Sanger

o

Synthesis Sequencing (Third Generation Sequencing)




Gene
Expression

Analysis

o

Northern Blotting



Screening for mRNA
coding for protein of interest at different parts/timings of the metabolic pathway

/ developmental stages



Southern
-
blot similarity (Electrophoresis + Hybridization with Radioactive probe)

o

Western Blot



Screening for protein of interest at different parts/ti
mes of metabolic pathway / developmental stages



Amino
-
acid sequence detection via hybridization with probes

o

Reverse transcriptase
-
polymerase chain reaction



cDNA synthesis from mRNA present at time of interest during metabolic pathway / developmental stages




PRC amplification using gene specific primers



Gel electrophoresis indicates presence of mRNA coming from gene only when protein is active at the time of sampling

o

In situ hybridization



Probes dyed will show active mRNA elements in cells that express
gene.

o

System Approach



Developmental tracking of gene expression



Gene to gene relationships (epistasis, polygenic traits, pleitropy)



Gene to environment interactions (epistasis, signal transduction))



cDNA vs. DNA comparison for identification of active gene
s



Above vs. gene mapping (Linkage; Physical; Cytogenic; Sequencing; Coiling)

o

DNA Microarrays



mRNA isolation



cDNA synthesis with fluorescent/dyed nucleotides



Hybridization with fragments from organisms genes



Different gene for each spot



Active genes will be

died



Can test multiple genes at once



Gene Function Analysis

o

In vitro mutagenesis

o

RNAi via siRNA

o

Genome
-
wide studies

o

Single Nucleotide Polymorphism

o

Restrict
ion fragment length polymorphism


Cloaning & Stem Cell Research



Basics

o

DNA
vs. Cell

vs. Organism
Cloning

o

Cloning & Genomic Equivalence After Differentiation

o

Totipotent / Stem Cells



Plant Cloning

o

Seeds

o

Cutting



Animal Cloning

o

In Vitro Fertilization

o

Dnucleation / UV Nuclear Sterilization

o

Nuclear Transplantation

o

Embryonic Implantation

o

Differentiation &
Limits to Nuclear Transplantation

o

Cell cycle arrest & dedifferentiation

o

Clone Phenotype Differences (Epigenetics & Chromosomal/Gene Activation/Deactivation)

o

Stem Cells



Embryonic: Undifferentiated (Totipotent)



Adult: Semi differentiated (Muscle Base; Bone M
arrow; Umbilical cord) (Pluripotent)



Reversed: Dedifferentiated (Induced Pluripotent)

o

Cloning by Blastula Separation



Applications

o

Trait proliferation

o

Specimen Preservation/Conservation

o

Species Recovery

o

Therapeutic Cloning

o

Cell Reversal as Disease Treatment

and Understanding



Limitations

o

Ethics

o

Underdevelopment (Nuclear Deactivation)

o

Biodiversity


Biotechnology Applications




Medical

o

Identification of Genetic Disorders by Gene Sequencing & Analysis


o

Embryonic defect detection (amniocentesis)

o

Study of Defective

Genes

o

Genome Studies & Endemics

o

Disease Profiling

o

Personalized Medicine (Based on Gene/Expression Patterns)

o

Cell Targeting

o

Gene Therapy



Vector (Retroviral / Manual Cell Implantation) modification of genetic code



Present: Stem Cell Application (Bone Marrow
) vs. Future: System wide gene therapy



Embryonic genetic engineering



Limitations:



Specificity of vectors



Gene expression control



Pleitropy / Epistasis



Eugenics



Ethics



Pharmaceutical Products

o

Specific cell delivery

o

Small molecular synthesis

o

Protein
Production

o

Transgenic Animals & “Pharm”
ing



Type / need matching



Purity testing



Forensics

o

Genetic Profiling



Extraction


Cleaving


Cloning (usually by PCR)


Eletrophoresis



Short tandem repeats



Crime Scene Investigation & Paternity



Environmental Cleanup



Agricultural

o

Organism Hybridization

o

Artificial Selection

o

Cloning good crops (Easier to dedifferentiate plants

o

Genetic engineering



Transgenic organisms for protection / yield



Gene modification / therapy



Common Vector: Ti plasmid in

Agrobacterium tumefaciens

o

Food modifications & Human Interactions