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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