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9| DNA-Based Information Technologies - Coggle Diagram
9| DNA-Based Information Technologies
DNA Cloning
DNA cloning:
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 gene into the vector.
Let the host produce multiple copies of recombinant DNA.
開始動作 :
Step 1 Generate Recombinant Vector
Restriction Endonucleases
Cleave DNA phosphodiester bonds at specific sequences • Common in bacteria
– eliminates infectious viral DNA • Some make staggered cuts.
– sticky ends
• Some make straight cuts.
– blunt ends
• Large number are known
– commercially available
– well documented: REBASE
–
http://rebase.neb.com/rebase/rebase.html
DNA Ligase
Enzyme that covalently joins two DNA fragments
Normally function in DNA repair
– Human DNA ligase uses ATP.
– Bacterial DNA ligase uses NAD.
Step 2 Introduce DNA into Organism
Antibiotic Selection
Antibiotics, such as penicillin and ampicillin, kill bacteria. • Plasmids can carry genes that give a host bacterium a
resistance against antibiotics. • Allows growth (selection) of bacteria that have taken
up the plasmid
Identification of Empty Plasmids
Cloning Vectors
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
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: BAC
Cloning Vectors: YAC
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 …
Learning goals:
DNA cloning techniques
Expression of recombinant proteins
DNA analysis methods
DNA microarray technology
SNPs:
Single Nucleotide Polymorphisms (SNPs) Can Distinguish Human Populations
之所以可以區別人種的原因
Tag SNPs
有三種
(b)haplotype:把(a)都交出來串在一起
(c)tag SNPs:(b)中不會每個位置都檢查，進一步挑出tag
(a)SNPs: 完整chromosome上某些allel
之所以要區別人種
跟藥物的治療反應、疾病的發生有關
這是一種研究方法：SNPs對人類or動物有什麼影響？
以上(a)(b)(c)的挑選過程
Expression of Cloned Genes
clone進去的東西我們想要他過度表現 但是怎麼知道有沒有過度表達
介紹
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:(expression vectors跟cloning vector之間的差異)
promoter sequences
operator sequences
code for ribosome-binding site
transcription termination sequences
Typical Expression Vector
檢測Purification of Recombinant Genes
在protein上插tag並加上相對應的ligand就會發光、並把涵有target protein和tag的fusion protein用ligand釣出來
（前面的Tag會發光）Fluorescence Can Be Used to Determine Protein Location In Vivo
Green fluorescent protein (GFP)
visualize with a fluorescent microscope
use recombinant DNA technologies to attach GFP to
protein of interest
Immunofluorescence免疫螢光反應
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
DNA Microarrays(設計晶片短時間比對十萬筆資料) Show Differences in Gene Expression
介紹
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.
晶片製作方式
Two-Color DNA Microarray Analysis on “Genome Chips”
Applications
原因DNA microarrays allow simultaneous screening of many thousands of genes: high-throughput screening.(也叫dirty data要用方法辨識)
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?
考：可以轉錄成蛋白質的基因佔比有多少？Genome基因組中的sequence序列types
Polymerase Chain Reaction (PCR)
介紹
• Mix together.(原料必記)
– target DNA
．– primers (oligonucleotides complementary to target) .
nucleotides: dATP, dCTP, dGTP, dTTP
–– thermostable DNA polymerase
其實還有buffer、離子 影響pH值
• Place the mixture into a thermocycler.溫度是重要因素
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.
用途
Used to amplify DNA in the test tube
can amplify regions of interest (genes) within linear DNA
can amplify complete circular plasmids
(圖解)開始動作‘
Repeat Steps 1–3 Many Times
After 25 cycles, DNA has been amplified about 106 fold.
P完蛋白質DNA之後就要Separation of DNA by Electrophoresis電泳
Negatively charged DNA migrates to the anode in the
presence of an electric field.
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-protein interaction studies
應用Applications and Adaptations of PCR
Applications
Cloning
use flanking primers to replicate and amplify a specific segment of DNA
Site-directed mutagenesis (oligonucleotide-directed)
後續介紹
use “sandwich” primers to make small changes to the segment of DNA and then amplify the altered DNA
介紹
Understanding the function of proteins often requires
• To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene.
• Site-directed mutagenesis usually relies on chemically synthesized mutated primers that are incorporated into newly synthesized DNA.
• Mutated plasmids are always sequenced to confirm that the desired (and only the desired) mutation is present
DNA fingerprinting
記住STR(short tandem repeats)
use primers for specific chromosomal locations to amplify and determine
number of short tandem repeats for purposes of identification
介紹
Humans have short sequences that repeat next to each other.
Differences in the number of repeats cause variations in the
length of fragments that form when a sample is subjected to PCR using a primer specific for that region.
Fragment sizes can be determined by using a capillary gel.
Multiple STR locations exist in the human genome.
Allows matching “suspect” samples to known individuals
Thirteen well-studied locations are used in identifications.
based on number of alleles seen at each location, with
misidentification less than 1 in 1018 (when good data are obtained)
P.26每個人有的指紋不同
Adaptations
Reverse transcriptase PCR (RT-PCR)
used to convert RNA code to DNA and amplify specific segments
目的Eukaryotic Gene Expression in Bacteria
真核Eukaryotic genes have一堆introns 無法人工去除的垃圾
解方mRNA is intron-free genetic material, as the codons
have already been spliced out從mRNA反轉錄回來因為mRNA是DNA自己已經轉錄過、去除intron, 而把mRNA反轉錄後會產生互補的cDNA
過程說明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.
圖解
Quantitative PCR (Q-PCR)
used to show quantitative differences in gene levels
介紹p.27用DNA中螢光物質測數量
