Download DNA Sequencing

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
restriction
enzyme (cut)
A A restriction
enzyme recognizes
a 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.
mix
B Researchers use
restriction enzymes to
cut DNA from different
sources into fragments.
Fragments with identical
sticky ends are mixed
together.
DNA ligase
(paste)
C Matching sticky
ends of different
fragments base-pair
with each other,
regardless of the
source of the DNA.
D DNA ligase joins
the fragments of
DNA where they
overlap. Molecules
of recombinant DNA
are the result.
Stepped Art
Fig. 16-2, p. 242
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
enzyme
A A restriction enzyme
cuts a specific base
sequence everywhere
it occurs in DNA.
B The same
enzyme cuts the
same sequence
in plasmid DNA.
C The DNA
fragments
have sticky ends.
D The plasmid DNA
also has sticky ends.
E The DNA
fragments
and the cut
plasmid are
mixed. The
sticky ends
of different
fragments
that basepair are
bonded by
DNA ligase.
F The result?
Recombinant
plasmids that
carry foreign DNA.
These plasmids are
introduced into host
cells, which divide
to form clones.
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
A Individual bacterial cells from a DNA library are
spread over the surface of a solid growth medium.
The cells divide repeatedly and form colonies—clusters
of millions of genetically identical daughter cells.
B A piece of special paper pressed onto the surface of
the growth medium will bind some cells from each colony.
C The paper is soaked in a solution that ruptures the
cells and releases their DNA. The DNA clings to the
paper in spots mirroring the distribution of colonies.
D A probe is added to the liquid bathing the paper.
The probe hybridizes with (sticks to) only the spots of
DNA that contain complementary base sequences.
E The bound probe makes a spot. Here, one radioactive
spot darkens x-ray film. The position of the spot on the film
is compared to the positions of all the original bacterial
colonies. Cells from the colony that made the spot are
cultured, and the DNA they contain is harvested.
Fig. 16-5, p. 244
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 basepair 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
PCR
A DNA template (purple) is mixed with
primers (red), free nucleotides, and
heat-tolerant Taq DNA polymerase.
B When the mixture is heated, DNA strands
separate. When it is cooled, some primers
hydrogen-bond to the template DNA.
C Taq polymerase uses the primers to
initiate synthesis, and complementary strands
of DNA form. The first round of PCR is now
complete.
D The mixture is heated again, and all
of the DNA separates into single strands.
When the mixture is cooled, some of the
primers hydrogenbond to the 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 of
the template DNA.
Fig. 16-6, p. 245
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
A The fragment of DNA
to be sequenced is mixed
with a primer, DNA polymerase, and nucleotides.
The mixture also includes
the four dideoxynucleotides
labeled with four different
colored pigments.
B The polymerase
uses the DNA as a template to synthesize new
strands again and again.
Synthesis of each new
strand stops when a dideoxynucleotideis added.
C At the end of the reaction,
there are many truncated
copies of the DNA
template in the mixture.
E A computer detects
and records the color of
each band on the gel. The
order of colors of the bands
represents the sequence of
the template DNA.
D An electrophoresis gel separates the fragments
into bands according
to length. All fragments
in each band end with the
same dideoxynucleotide;
thus, each band is the color
of that dideoxynucleotide.
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-andloop 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
A An A.
tumefaciens
bacterium
contains
a Ti plasmid
that has been
engineered
to carry a
foreign gene.
B The bacterium
infects a plant
cell and transfers
the Ti plasmid into
it. The plasmid DNA
becomes integrated
into one of the plant
cell’s chromosomes.
C The plant
D Transgenic plants
cell divides. Its
descendant cells
form an embryo,
which may
develop into
a mature plant
that can express
the foreign gene.
E A young
tobacco
plant visibly
expressing
a foreign
gene.
Fig. 16-12, p. 251
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 (SCIDX1) – 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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