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DNA-Based Information Technologies - Coggle Diagram
DNA-Based Information Technologies
Recombinant DNA
molecular cloning of genes
overexpression of proteins
transgenic food, animals
DNA Cloning
Steps
Cut the source DNA at the boundaries of the gene.
Select a carrier(vector).
Insert the gene into the vector.
Insert the recombinant vector into host cell.
Propagation(cloning)
Restriction Endonucleases
Cleave DNA phosphodiester bonds
staggered cuts
(sticky ends)(尖端)
EcoRl
straight cuts
(blunt ends)(平的)
Pvull
DNA Ligase
DNA repair
Human DNA ligase uses ATP.
Bacterial DNA ligase uses NAD.
Antibiotic Selection
是否篩進去
Cloning Vectors
Plasmids
carry antibiotic resistance genes
cloning of DNA up to 15,000 bp
bacterial artificial chromosome (BAC)
藍白篩(白)
yeast artificial chromosome (YAC)
Linear chromosome
Polymerase Chain Reaction (PCR)
amplify DNA in the test tube
linear DNA or circular plasmid
Mix together
target DNA
primers (oligonucleotides complementary to target)
nucleotides: dNTP
thermostable DNA polymerase
Buffer
Place the mixture into a thermocycler.
Melt DNA at 95^C
Cool separated strands to about 50-60^C.
Primers anneal to the target.
5’-3’direction
Applications
Cloning
Site-directed mutagenesis (oligonucleotide-directed)
Understanding the function of proteins
DNA fingerprinting
short tandem repeats (STR)
13組
Adaptations
Reverse transcriptase PCR (RT-PCR)
Quantitative PCR (Q-PCR)
CT值愈小
量愈多
Eukaryotic Gene Expression in Bacteria
exons: coding regions
introns: noncoding regions
Bacteria cannot splice introns out of coding DNA.
mRNA is intron-free genetic material, as the codons have already been spliced out.
Construction of cDNA
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.
reverse transcriptase
Expression of Cloned Genes differ from cloning vectors by having:
promoter sequences
operator sequences
code for ribosome-binding site
transcription termination sequences
Purification of Recombinant Genes
Recombinant proteins can be tagged for purification.
Fluorescence
Green fluorescent protein (GFP)
Immunofluorescence
一抗 二抗
DNA Microarrays
mRNA or cDNA from different samples are differentially tagged.
Analysis on the same chip shows differences.
high-throughput screening
Genome-wide genotyping
Tissue-specific gene expression
Mutational analysis
Protein-coding genes(1.5%)
Single Nucleotide Polymorphisms (SNPs) Can Distinguish Human Populations
單倍數基因型