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DNA-Based Information Technologies - Coggle Diagram
DNA-Based Information Technologies
Recombinant DNA
do not occur together in the nature
Basis of much of the modern molecular biology
DNA Cloning
Creation of identical copies of a piece of DNA (gene)
從源生物中分離出特定基因，並在目標生物體中放大它。
Basic steps
1.Cut the source DNA at the boundaries of the gene.
2.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
staggered cuts
sticky ends
straight cuts
blunt ends
DNA Ligase
共價連接兩個 DNA 片段的酶
Normally function in DNA repair
Human DNA ligase uses ATP.
Bacterial DNA ligase uses NAD.
Antibiotic Selection
kill bacteria
質粒可以攜帶使宿主細菌對抗生素產生抗性的基因。
Identification of Empty Plasmids
Cloning Vectors
Plasmids
環狀 DNA 分子與 細菌基因組DNA
replicate autonomously
carry antibiotic resistance genes
cloning of DNA up to 15,000 bp
clone whole chromosomes
up to 300,000 bp
bacterial artificial chromosome (BAC)
use in bacteria
very similar to using plasmids
lacZ gene
required for the production of the enzyme β-galactosidase
X-gal
a substrate for β- galactosidase that yields a blue product
active β-galactosidase
blue
without β-galactosidase activity
white
yeast artificial chromosome (YAC)
use in yeast
the simplest eukaryote for DNA recombination.
linear chromosomes
unstable in vitro
YAC 是圓形的，以適應快速復制和穩定存儲，但有一個可移動的部分，可以線性化產物以進行轉換。
Polymerase Chain Reaction (PCR)
amplify DNA in the test tube
Mix together
target DNA
primers(oligonucleotides complementary to target)
nucleotides
dATP, dCTP, dGTP, dTTP
thermostable DNA polymerase
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
Applications
Cloning
Site-directed mutagenesis
可檢測一個protein上某個點位的功能或構造
To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene.
DNA fingerprinting
人類具有彼此相鄰重複的短序列
short tandem repeats (STR)
重複次數的差異會導致片段長度的變化，這些片段在使用特定於該區域的引物對樣品進行 PCR 時形成。
Fragment sizes can be determined by using a capillary gel
人類基因組中存在多個 STR 位置
Thirteen well-studied locations are used in identifications
Adaptations
Reverse transcriptase PCR (RT-PCR)
Quantitative PCR (Q-PCR)
沒打開
不發光
打開
發光
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
Practical use
DNA analysis
DNA purification
DNA-proteininteractionstudies
Eukaryotic Gene Expression in Bacteria
A eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium.
Eukaryotic genes have
exons
coding regions
introns
noncoding regions
Introns in eukaryotic genes pose problems.
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
mRNA can be extracted from eukaryotic cells.
All mRNA molecules have a poly(A) tail.
有助於純化mRNA
serves as a universal template
catalyzed by the reverse transcriptase
mRNA 在鹼性的狀態下被降解
Expression of Cloned Genes
expression vectors
differ from cloning vectors by having
promoter sequences
code for ribosome-binding site
operator sequences
transcription termination sequences
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 of interest
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.
DNA Microarrays Show Differences in Gene Expression
Analysis on the same chip shows differences.
Microarray chips contain fragments from genes
Applications
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?