studying and manipulating genomes
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Studying and Manipulating Genomes. Chapter 16 Hsueh-Fen Juan Oct 30, 2012. 16.1 Cloning DNA. Researchers cut up DNA from different sources, then paste the resulting fragments together Cloning vectors can carry foreign DNA into host cells. Cut and Paste. Restriction enzymes - PowerPoint PPT PresentationTRANSCRIPT
Studying and Manipulating Genomes
Chapter 16
Hsueh-Fen Juan
Oct 30, 2012
16.1 Cloning DNA
Researchers cut up DNA from different sources, then paste the resulting fragments together
Cloning vectors can carry foreign DNA into host cells
Cut and Paste
Restriction enzymes• Bacterial enzymes that cut DNA wherever a
specific nucleotide sequence occurs• 之所以叫限制酶,是因為它的發現來自於,科學家
發現有的細菌不怕噬菌體,因為噬菌體 DNA 一注入,這種細菌的酶會馬上去分解此噬菌體 DNA 的特定序列部位,「限制」噬菌體的感染,故名限制酶。
Single-stranded DNA tails produced by the same restriction enzyme base-pair together• DNA ligase bonds “sticky ends” together
Recombinant DNA• Composed of DNA from two or more organisms
Animation: Base-pairing of DNA fragments
Animation: Restriction enzymes
Making Recombinant DNA
Fig. 16-2, p. 242
A A restriction enzyme recognizesa specific base sequence in DNA (red boxes, 6個非 4個 ). For this and many other enzymes, the sequence is the same in the 5 to 3 direction on both strands.
B Researchers use restriction enzymes to cut DNA from differentsources into fragments.Fragments with identical sticky ends are mixed together.
restrictionenzyme (cut)
C Matching sticky ends of different fragments base-pair with each other,regardless of the source of the DNA.
mix
D DNA ligase joins the fragments of DNA where they overlap. Molecules of recombinant DNA are the result.
DNA ligase(paste)
Stepped Art
DNA Cloning
DNA cut into fragments by restriction enzymes is inserted into cloning vectors (plasmids) cut with the same enzyme
Cloning vectors with foreign DNA are placed in host cells, which divide and produce many clones, each with a copy of the foreign DNA
Cloning Vectors
DNA Cloning
B The same enzyme cuts the same sequence in plasmid DNA.
C The DNA fragmentshave sticky ends.
E The DNAfragmentsand the cutplasmid aremixed. Thesticky endsof differentfragmentsthat base-pair arebonded byDNA ligase.
enzyme
F The result? Recombinantplasmids that carry foreign DNA. These plasmids areintroduced into host cells, which divide to form clones.
A A restriction enzymecuts a specific basesequence everywhereit occurs in DNA.
D The plasmid DNAalso has sticky ends.
Animation: Formation of recombinant DNA
cDNA Cloning
Complementary DNA (cDNA)• DNA made from an mRNA template• 在真核即「只包含外顯子部分的 DNA 」
Reverse transcriptase transcribes mRNA to DNA, forming a hybrid molecule• DNA polymerase builds a double-stranded DNA
molecule that can be cloned
cDNA Cloning
16.2 From Haystacks to Needles
DNA libraries and the polymerase chain reaction (PCR) help researchers isolate particular DNA fragments
DNA Libraries
Genome• The entire set of genetic material of an organism
DNA libraries are sets of cells containing various cloned DNA fragments• Genomic libraries (all DNA in a genome)• cDNA libraries (all active genes in a cell)
Animation: How to make cDNA
Probes
Probe• A fragment of DNA labeled with a tracer• Used to find a specific clone carrying DNA of
interest in a library of many clones
Nucleic acid hybridization• Base pairing between DNA from different sources• A probe hybridizes with the targeted gene
Nucleic Acid Hybridization using a Radioactive Probe
Fig. 16-5, p. 244
D A probe is added to the liquid bathing the paper.The probe hybridizes with (sticks to) only the spots ofDNA that contain complementary base sequences.
E The bound probe makes a spot. Here, one radioactivespot darkens x-ray film. The position of the spot on the filmis compared to the positions of all the original bacterialcolonies. Cells from the colony that made the spot arecultured, and the DNA they contain is harvested.
B A piece of special paper pressed onto the surface ofthe growth medium will bind some cells from each colony.
C The paper is soaked in a solution that ruptures thecells and releases their DNA. The DNA clings to thepaper in spots mirroring the distribution of colonies.
A Individual bacterial cells from a DNA library arespread over the surface of a solid growth medium.The cells divide repeatedly and form colonies—clustersof millions of genetically identical daughter cells.
Animation: Use of a radioactive probe
Big-Time Amplification: PCR
Polymerase chain reaction (PCR)• A cycled reaction that uses a heat-tolerant form
of DNA polymerase (Taq polymerase) to produce billions of copies of a DNA fragment
• 因為 PCR 過程要重複加溫降溫,而一般的 DNA聚合酶在高溫 ( 能解開螺旋的溫度 ) 時通常會被破壞,而一種耐高溫細菌 (Thermus aquaticus, aka Taq) 的 DNA 聚合酶就很適合用在這裡
PCR
DNA to be copied is mixed with DNA polymerase, nucleotides and primers that base-pair with certain DNA sequences
Cycles of high and low temperatures break and reform hydrogen bonds between DNA strands, doubling the amount of DNA in each cycle
Fig. 16-6, p. 245
A DNA template (purple) is mixed with primers (red), free nucleotides, andheat-tolerant Taq DNA polymerase.
D The mixture is heated again, and allof the DNA separates into single strands.When the mixture is cooled, some of theprimers hydrogenbond to the DNA.
C Taq polymerase uses the primers toinitiate synthesis, and complementary strandsof DNA form. The first round of PCR is nowcomplete.
B When the mixture is heated, DNA strandsseparate. When it is cooled, some primershydrogen-bond to the template DNA.
E Taq polymerase uses the primers to initiate DNA synthesis, and complementary strands of DNA form. The second round of PCR is complete.
Each round can double the number of DNA molecules. After 30 rounds, the mixture contains huge numbers of DNA fragments, all copies ofthe template DNA.
PCR
Animation: Polymerase chain reaction (PCR)
16.3 DNA Sequencing
DNA sequencing reveals the order of nucleotide bases in a fragment of DNA
DNA Sequencing
DNA is synthesized with normal nucleotides and dideoxynucleotides (雙去氧核醣核苷酸 ) tagged with different colors • When a tagged base is added, DNA synthesis
stops; fragments of all lengths are made• 雙去氧核醣核苷酸的 3’ 沒有氧,因此無法讓下一個
核苷酸的 5’ 接上來,因此會停在那裏 Electrophoresis ( 電泳 ) separates the fragments
of DNA, each ending with a tagged base, by length• Order of colored bases is the sequence of DNA
Tagged Dideoxynucleotides
Each base is labeled with a different color
DNA Sequencing
E A computer detectsand records the color ofeach band on the gel. Theorder of colors of the bandsrepresents the sequence ofthe template DNA.
C At the end of the reaction,there are many truncatedcopies of the DNAtemplate in the mixture.
D An electro-phoresis gel sepa-
rates the fragmentsinto bands according
to length. All fragmentsin each band end with thesame dideoxynucleotide;
thus, each band is the colorof that dideoxynucleotide.
A The fragment of DNAto be sequenced is mixedwith a primer, DNA poly-merase, and nucleotides.The mixture also includesthe four dideoxynucleotideslabeled with four different colored pigments.
B The polymeraseuses the DNA as a tem-plate to synthesize newstrands again and again.Synthesis of each newstrand stops when a dide-oxynucleotideis added.
Animation: Automated DNA sequencing
Next Generation Sequencing (NGS)
Trends in Genetics 2008, 24 (3): 142–149.
16.4 DNA Fingerprinting
One individual can be distinguished from all others on the basis of DNA fingerprints
DNA Fingerprints
DNA fingerprint• A unique array of DNA sequences used to
identify individuals Short tandem repeats (STRs) 短縱列重複序列• 人類約有 99% 基因相同,剩下 1% 差異很大• Many copies of the same 2- to 10-base-pair
sequences in a series along a chromosome 是很短且重複排在一起的片段
• Types and numbers of STRs vary greatly among individuals
Creating DNA Fingerprints
PCR is used to amplify DNA from regions of several chromosomes that have STRs
Electrophoresis is used to separate the fragments and create a unique DNA fingerprint
DNA fingerprints have many applications• Legal cases, forensics, population studies
DNA Fingerprints: A Forensics Case
Animation: DNA fingerprinting
16.5 Studying Genomes
Comparing the sequence of our genome with that of other species is giving us insights into how the human body works
The Human Genome Project
Automated DNA sequencing and PCR allowed human genome projects to sequence the 3 billion bases in the human genome
28,976 genes have been identified, but not all of their products or functions are known
Sequencing the Human Genome
Genomics
Genomics: The study of genomes• Structural genomics 專注於基因所扁馬出來之
蛋白質的三度空間立體結構• Comparative genomics 專注於比較不同物種間
的基因差異 Analysis of the human genome yields new
information about genes and how they work• Applications in medicine and other fields• Example: APOA5 mutations and triglycerides
DNA Chips
DNA chips• Microarrays of many different DNA samples
arranged on a glass plate• Used to compare patterns of gene expression
among cells of different types or under different conditions
• May be used to screen for genetic abnormalities, pathogens, or cancer
DNA Chips and Gene Expression
Principle of cDNA microarrays
Principle of cDNA microarrays
Principle of oligonucleotide arrays
Construction of Oligonucleotide Arrays
Construction of Oligonucleotide Arrays
Microarray Images Produced with a Pin-and-loop Arrayer
16.6 Genetic Engineering
Genetic engineering• A laboratory process by which deliberate
changes are introduced into an individual’s genome
The most common genetically modified organisms are bacteria and yeast• Used in research, medicine, and industry• Example: To produce human insulin
GMOs
Genetically modified organisms (GMOs)• Individuals containing modified genes from the
same species or a different species
Transgenic organisms• Individuals containing genes transferred from a
different species (also GMOs)• Example: Bacteria with jellyfish genes
Transgenic Organisms
Bacteria with jellyfish genes
16.7 Designer Plants
Genetically engineered crop plants are widespread in the United States
The Ti Plasmid
Ti plasmid• Plasmid of bacteria Agrobacterium tumefaciens• Contains tumor-inducing (Ti) genes• Used as a vector to transfer foreign or modified
genes into plants, including some food crops• 注意,會先移除引起腫瘤的基因再加上想要的基因
Ti Plasmid Transfer
Fig. 16-12, p. 251
E A young tobaccoplant visibly expressinga foreign gene.
A An A. tumefaciensbacterium containsa Ti plasmid that has been engineered to carry a foreign gene.
B The bacterium infects a plant cell and transfersthe Ti plasmid into it. The plasmid DNA becomes integrated into one of the plant cell’s chromosomes.
C The plant cell divides. Its descendant cells form an embryo, which may develop into a mature plant that can express the foreign gene.
D Transgenic plants
Animation: Gene transfer using a Ti plasmid
Animation: Transferring genes into plants
Genetically Engineered Plants
Crop plants are genetically modified to produce more food at lower cost• Resistance to disease or herbicides• Increased yield• Plants that make pesticides ( 農藥 ) (Bt protein)• Drought resistance
Some Genetically Modified Plants
16.8 Biotech Barnyards
Animals that would be impossible to produce by traditional breeding methods are being created by genetic engineering
Genetically engineered animals are used in research, medicine, and industry
Of Mice and Men
1982: The first transgenic animals – mice with genes for rat growth hormone ( 利用 plasmid)
Examples of Transgenic Animals
Genetically modified animals are used as models of many human diseases• Mice used in knockout experiments
Genetically modified animals make proteins with medical and industrial applications• Goats and rabbits that make human proteins• Farms animals with desirable characteristics
Some Genetically Modified Animals
Knockout Cells and Organ Factories
Transgenic pigs with human proteins are a potential source of organs and tissues for transplants in humans• May prevent rejection by immune system• 自我辨識是看細胞膜上的 glycoprotein( 醣蛋白 )
Xenotransplantation• Transplantation of a tissue or organ from one
species to another• 有潛在風險,例如移植豬身上的基改組織、器官到
人身上,有可能打破病毒的 species barrier ,產生感染人類的新病毒,造成毀滅性危機
16.9 Safety Issues
The first transfer of foreign DNA into bacteria ignited an ongoing debate about potential dangers of transgenic organisms that may enter the environment
Into the Unknown
Early questions about new technologies:• Could genetic recombination in laboratories
produce a new superpathogen?• What if new forms escaped into the environment
and transformed other organisms?
Safety guidelines for transgenic research were adopted in the US and other countries
16.10 Modified Humans?
We as a society continue to work our way through the ethical implications of applying new DNA technologies
The manipulation of individual genomes continues even as we are weighing the risks and benefits of this research
Gene Therapy
Gene therapy• Transfer of recombinant DNA into body cells to
correct a genetic defect or treat a disease• Viral vectors or lipid clusters insert an unmutated
gene into an individual’s chromosomes• Examples: Cystic fibrosis, SCID-X1
Getting Better
1998: A viral vector was used to insert unmutated IL2RG genes into boys with severe combined immunodeficiency disease (SCID-X1) – most recovered immune function
Getting Worse
No one can predict where a virus-injected gene will insert into a chromosome – several boys from the SCID-X1 study developed cancer
In other studies, severe allergic reactions to the viral vector itself have resulted in death
Getting Perfect
Eugenic engineering (優生學 )• Engineering humans for particular desirable traits,
not associated with treatment of disorders
Many questions must be answered about the ethics and consequences of manipulating the human genome
ABC video: Glow-in-the-Dark Pigs
Synthetic Biology
2012.12.10-12/12 http://u.csie.org/IECA2012 台大物理館國際會議廳
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