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DNA- Based information technologies - Coggle Diagram
DNA- Based information technologies
Recombinant DNA
Artificially created DNA
DNA Cloning
Purpose
Creation of identical copies of a piece of DNA (gene)
Isolate a specific gene
amplify it in the target organism
Basic steps
1 Cut the source DNA at the boundaries of the gene(restriction endonuclease)
2.Select a suitable carrier DNA (vector)
Generate vector
Cleave vector with restriction endonuclease
Combine with DNA fragment with DNA ligase
Insert the gene into the vector.
4.insert into the host cell
Let the host produce multiple copies of recombinant DNA
Restriction endonuclease
Cleave DNA phosphodiester bonds at specific sequences
Common in bacteria
– eliminates infectious viral DNA
DNA ligase
DNA repair
Human DNA ligase uses ATP.
Bacterial DNA ligase uses NAD.
Antibiotics selection
Also use restriction endonuclease and DNA ligase
Cloning vectors
Plasmids
Can replicate autonomously
allow cloning of DNA up to 15000bp
BAC(bacteria)
Clone whole chromosomes
a colorimetric approach using X-gal is employed to select for colonies containing the chromosomal insert.
YAC(yeast)
They have linear chromosomes with telomere ends
YAC is circular to accommodate quick replication and stable storage
has a removable segment that linearizes the product for transformation.
PCR
Amplify DNA in the test tube
Specific sequence(gene)
complete circular plasmids
Mixed with
Target DNA
Primer(oligonucleotides)
Nucleotides
DNA polymerase
5’→ 3’DNA synthesis
Applications
Cloning (flanking primers)
Site-directed mutagenesis 希望其中一點突變
change the nucleotide(s) in the coding DNA and express the mutated gene.
Chemically synthesized mutated primers
DNA 定序 確認是否完成
Steps: 1. Recombinant vector cleave with restriction endonuclease
Insert synthetic DNA segment containing mutation
DNA fingerprinting (STR)
Short sequences that repeat next to each other
Differences in the number of repeats cause variations in the length of fragments
Fragment sizes can be determined by using a capillary gel
Thirteen well-studied locations are used in identifications.
Oligonucleotide-directed mutagenesis
Step 1. Denature plasmid and Anneal oligonucleotide primers with mutation
Step2. Use DNA polymerase to extend and incorporate the mutagenic primers
Step3. Digest nonmutated parental DNA template （with methylation-specific nuclease ）
Step4. Anneal newly synthesized strand
Step 5.transform dsDNA into cells.Cell repairs nicks in mutated plasmid
Adaptation
RT-PCR: RNA ->cDNA(only amplify extron)
Q-PCR Quantitative PCR
show quantitative differences in gene levels
Flourophore
Separation of DNA by Electrophoresis 電泳
Negatively charged DNA migrates to the anode
Agarose gel hinders the mobility of DNA molecules
Motibility
Size
Shape(compact faster)
Use
DNA analysis
DNA purification
DNA-protein interaction studies
Construction of cDNA
mRNA ft. Reverse transcriptase turn into cDNA
All mRNA molecules have a poly(A) tail
helps in purification of mRNA
serves as a universal template
Expression of Cloned Genes
Expression vectors vs. cloning vectors
operator sequences
code for ribosome-binding site
promoter sequences
transcriptionterminationsequences
Expression vectors have:
Purification of Recombinant Genes
Recombinant proteins can be tagged for purification
Fluorescence Can Be Used
to Determine Protein Location In Vivo:
Green fluorescent protein (GFP)
use recombinant DNA technologies to attach GFP to protein
visualize with a fluorescent microscope
Visualization of Protein Location from a GFP–Tagged cDNA Library
Immunofluorescence
tag protein with primary antibody
secondary antibody containing fluorescent tag
the primary antibody detecting the epitope can be fluorescently labeled
DNA Microarrays
mRNA or cDNA from different samples are differentially tagged.
Photolitographic Synthesis of DNA- Containing
Microchips
Two-Color DNA Microarray Analysis on
“Genome Chips”
Show Differences in Gene Expression
Application
High-throughput screening
Tissue-specific gene expression
Mutational analysis
Genome-wide genotyping
Single Nucleotide Polymorphisms (SNPs) Can Distinguish Human Populations
