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DNA-Based Information Technologies - Coggle Diagram
DNA-Based Information Technologies
Polymerase Chain Reaction (PCR)
General Steps of PCR
Repeat Steps 1–3 Many Times
Separation of DNA by Electrophoresis
Agarose gel hinders the mobility of DNA molecules.
Mobility depends on the size and the shape.
– small molecules faster
– compact molecules faster
Negatively charged DNA migrates to the anode in the
presence of an electric field.
Practical use
DNA analysis
DNA purification
DNA-protein interaction studies
Basic
Mix together.
target DNA
primers (oligonucleotides complementary to target)
nucleotides: dATP, dCTP, dGTP, dTTP
thermostable DNA polymerase
Used to amplify DNA in the test tube
– can amplify regions of interest (genes) within linear DNA
– can amplify complete circular plasmids
Place the mixture into a thermocycler.
Melt DNA at about 95°C.
Cool separated strands to about 50–60°C.
Primers anneal to the target.
Polymerase extends primers in the 5’→ 3’ direction.
After a round of elongation is done, repeat the steps.
Applications and Adaptations of PCR
DNA fingerprinting
– use primers for specific chromosomal locations to amplify and determine number of short tandem repeats for purposes of identification
Reverse transcriptase PCR (RT-PCR)
– used to convert RNA code to DNA and amplify specific segments
Site-directed mutagenesis (oligonucleotide-directed)
– use “sandwich” primers to make small changes to the segment of DNA and then amplify the altered DNA
Quantitative PCR (Q-PCR)
– used to show quantitative differences in gene levels
Cloning
– use flanking primers to replicate and amplify a specific segment of DNA
Recombinant DNA
Artificially created DNA that combines sequences that
do not occur together in the nature
Basis of much of the modern molecular biology
molecular cloning of genes
overexpression of proteins
transgenic food, animals …
Construction of cDNA
mRNA can be extracted from eukaryotic cells.
All mRNA molecules have a poly(A) tail.
– helps in purification of mRNA
– serves as a universal template
A DNA strand can be synthesized using mRNA as a template.
This is catalyzed by the reverse transcriptase.
The end result is a hybrid where the DNA strand is complementary to the mRNA.
The hybrid can be converted to duplex DNA, known as cDNA.
DNA Cloning
Basic
Creation of identical copies of a piece of DNA (gene)
Isolate a specific gene from the source organism and amplify it in the target organism.
Basic steps
Cut the source DNA at the boundaries of the gene.
Select a suitable carrier DNA (vector).
Insert the gene into the vector.
Insert the recombinant vector into host cell.
Let the host produce multiple copies of recombinant DNA.
Restriction Endonucleases
Cleave DNA phosphodiester bonds at specific sequences
Common in bacteria
– eliminates infectious viral DNA
Some make straight cuts.
– blunt ends
Large number are known
commercially available
well documented: REBASE
Some make staggered cuts.
– sticky ends
DNA Ligase
Enzyme that covalently joins two DNA fragments
– Normally function in DNA repair
– Human DNA ligase uses ATP.
– Bacterial DNA ligase uses NAD.
Antibiotic Selection
Antibiotics, such as penicillin and ampicillin, kill bacteria.
Plasmids can carry genes that give a host bacterium aresistance against antibiotics.
Allows growth (selection) of bacteria that have taken
up the plasmid
Antibiotic Selection
Identification of Empty Plasmids
Cloning Vectors
Cloning Vectors: Plasmid
Cloning Vectors: BAC
The process for cloning via BAC is very similar to using plasmids.
Often, a colorimetric approach using X-gal is employed to select for colonies containing the chromosomal insert.
lacZ gene (required for the production of the enzyme β-galactosidase)
Plates also contain X-gal, a substrate for β-galactosidase that yields a blue product.
Colonies with active β-galactosidase and hence no DNA insert in the BAC vector turn blue;colonies without β-galactosidase activity—and thus with the desired DNA inserts—are white.
To clone whole chromosomes
(up to 300,000 bp)
bacterial artificial chromosome (BAC)
• for use in bacteria
yeast artificial chromosome (YAC)
• for use in yeast
Cloning Vectors: YAC
Yeast are the simplest eukaryote for DNA recombination.
They have linear chromosomes with telomere ends, which can be unstable in vitro.
YAC is circular to accommodate quick replication and stable storage but has a removable segment that linearizes the product for transformation.
Plasmids
circular DNA molecules that are separate from the bacterial genomic DNA
can replicate autonomously
• origins of replication for use in bacteria and/or yeast
carry antibiotic resistance genes
allows cloning of DNA up to 15,000 bp
Eukaryotic Gene Expression in Bacteria
Introns in eukaryotic genes pose problems.
Bacteria cannot splice introns out of coding DNA.
Eukaryotic genes have:
exons: coding regions
introns: noncoding regions
mRNA is intron-free genetic material, as the codons have already been spliced out.
A eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium.
Expression of Cloned Genes
Typical Expression Vector
Purification of Recombinant Genes
Purification of natural proteins is difficult.
Recombinant proteins can be tagged for purification.
The tag binds to the affinity resin, binding the protein of interest to a purification column.
Basic
We want to study the protein product of the gene.
Special plasmids, called expression vectors, contain sequences that allow transcription of the inserted gene.
Expression vectors differ from cloning vectors by having:
promoter sequences
operator sequences
code for ribosome-binding site
transcription termination sequences
Purification of Recombinant Genes
Fluorescence
GFP–Tagged Protein Localization
Immunofluorscence
Immunofluorescence
tag protein with primary antibody and detect with secondary antibody containing fluorescent tag
Protein can also be fused to a short epitope, and the primary antibody detecting the epitope can be fluorescently labeled.
Visualization of Protein Location from a GFP–Tagged cDNA Library
Green fluorescent protein (GFP)
use recombinant DNA technologies to attach GFP to protein of interest
visualize with a fluorescent microscope
Microarrays
Human Genome Contains Many Different Sequence Types
DNA Microarrays Show Differences in Gene Expression
Microarray chips contain fragments from genes in the group to be analyzed.
– full genome of bacteria or yeast, or protein-encoding families from larger genomes
mRNA or cDNA from different samples are differentially tagged.
Analysis on the same chip shows differences.
Single Nucleotide Polymorphisms (SNPs) Can Distinguish Human Populations
DNA Microarrays: Applications
DNA microarrays allow simultaneous screening of many thousands of genes: high-throughput screening.
Genome-wide genotyping
– Which genes are present in this individual?
Tissue-specific gene expression
– Which genes are used to make proteins?
Mutational analysis
– Which genes have been mutated?
Two-Color DNA Microarray Analysis on “Genome Chips”
Photolitographic Synthesis of DNA- Containing Microchips