Download DNA Cloning

Genomics: The Technology
behind the Human
Genome Project
Shu-Ping Lin, Ph.D.
Institute of Biomedical Engineering
E-mail: [email protected]
Website: http://web.nchu.edu.tw/pweb/users/splin/
Electrophoresis
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Gel electrophoresis: separates nucleic acids or proteins on the basis of
size or electrical charge because molecule with net charge migrates in an electric
field creating DNA bands of the same length
Separating DNA fragments according to size produces: high-resolution separation of
DNA molecules
Velocity (v) of molecule depends on electric field strength (E), net charge on the
protein (z), frictional coefficient (f)
DNA Cloning
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Restriction enzymes (endonucleases):
in nature, these enzymes protect
bacteria from intruding DNA; they cut up the
DNA (restriction); very specific
Restriction site:
recognition sequence for a particular
restriction enzyme
Restriction fragments:
segments of DNA cut by restriction
enzymes in a reproducable way
Sticky end:
short extensions of restriction
fragments
DNA ligase:
enzyme that can join the sticky ends of
DNA fragments
Cloning vector:
DNA molecule that can carry foreign
DNA into a cell and replicate there (usually
bacterial plasmids)
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Recombinant
DNA
DNA coming from 2 different sources is fragmented using the same
restriction enzyme.
Matching sticky ends bring fragments from different sources together.
These pieces are covalently linked with the help of enzyme DNA ligase.
Insertion of DNA fragment into an open
circular DNA (an empty vector) and thus
the formation of cloning vector.
Bacterial Plasmids in Gene Cloning
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One basic cloning technique begins with the insertion of a
foreign gene into a bacterial plasmid (i.e. cloning vector).
Fig. 20.1
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Separation of cell
clones containing
insert DNA from
others
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Isolation of cloning vector
(bacterial plasmid) & genesource DNA (gene of
interest)
Insertion of gene-source
DNA into the cloning vector
using the same restriction
enzyme; bind the
fragmented DNA with DNA
ligase
Introduction of cloning
vector into cells
(transformation by bacterial
cells)
Cloning of cells (and
foreign genes)
Identification of cell clones
carrying the gene of interest
The process of making multiple copies
1. Plasmids and Cloning; 2. Recombinant
DNA; 3. Cloning Vectors; 4. Presenting
Vectors to Host Cells; 5. Selecting Cells
That Contain Insert DNA
Polymerase Chain Reaction (PCR)
Fig. 20.3
Copyright © 2002 Pearson Education, Inc.,
publishing as Benjamin Cummings
Making Multiple Copies
To study a particular gene, scientists needed to develop methods
to isolate only the small, well-defined, portion of a chromosome
containing the gene:
DNA cloning is the best method for preparing large quantities of
a particular gene or other DNA sequence.
Techniques for gene cloning enable scientists to prepare multiple
identical copies of gene-sized pieces of DNA.
 The original plasmid used to produce recombinant DNA is called a
cloning vector, which is a DNA molecule that can carry foreign DNA
into a cell and replicate there.
Bacteria are most commonly used as host cells for gene cloning
because DNA can be easily isolated and reintroduced into their cells.
Bacteria cultures also grow quickly, rapidly replicating the foreign
genes.
The source DNA comes from human tissue cells.
The source of the plasmid is typically E. coli.
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Polymerase Chain Reaction (PCR)
When the source of DNA is
scanty or impure, the
polymerase chain reaction
(PCR) is quicker and more
selective.  DNA is incubated
in a test tube with special
DNA polymerase, a supply
of nucleotides, and short
pieces of single-stranded DNA
as a primer.
Amplification of any piece of
DNA without cells (in vitro)
Materials: heat, DNA
polymerase, nucleotides,
single-stranded DNA primers
PCR can make billions of
copies of a targeted DNA
segment in a few hours.
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This is faster than cloning via
recombinant bacteria.
Fig. 20.7
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
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In PCR, a three-step
cycle--heating, cooling,
and replication -- brings
about a chain reaction that
produces an exponentially
growing population of
DNA molecules.
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The key to easy PCR
automation was the
discovery of an unusual
DNA polymerase, isolated
from bacteria living in hot
springs, which can withstand
the heat needed to separate
the DNA strands at the start
of each cycle. The enzyme is
called Taq.
Restriction Fragment Analysis
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Restriction fragment length polymorphisms (RFLPs)
Southern blotting: process that reveals sequences and
the RFLPs in a DNA sequence
DNA Fingerprinting
DNA Sequencing
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Sequence reading direction
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Determination of nucleotide sequences
Genomics: study of genomes based on
DNA sequences  Human Genome Project
DNA replication upon binding of analog
to base A results in DNA pieces with
varying lengths. Resulting fragments
loaded onto lane A of acrylamidesequencing gel
Facts About the Human Genome
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Diploid, 23 chromosome pairs
8% present in large recent duplications
Genes represent ~1.5-2% of genome sequence
Non-genic functional sequences = ??
Repetitive DNA = ~50%
3 x 109 bases
~30,000 genes
Chapter 15 Human Heredity by Michael Cummings © 2006 Brooks/Cole-Thomson Learning
We Share –
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Mapping human chromosome segments onto homologous segments
along mouse chromosomes  Numbers identifying sections of
mouse chromosomes refer to identity of human chromosomes
with similar sections
Practical DNA
Technology Uses
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DNA technology makes it possible to
clone genes for basic research and
commercial applications
Diagnosis of disease
Human gene therapy
Pharmaceutical products (vaccines)
Forensics
Animal husbandry (transgenic
organisms)
Genetic engineering in plants
Ethical concerns?
Cloning the first Human:
http://www.youtube.com/watch?v=Tw1CX6ku0NQ&feature=related
First test tube baby Louise Brown (1978) - Robert
Edwards Wins the Nobel Prize in Physiology or
Medicine for Pioneering In Vitro Fertilization (IVF)
http://www.youtube.com/watch?v=pqu8Y4XGFK4&NR=1
Plants and Animals with
Modified Genomes
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Dolly the sheep：
Name after~ & her unusual
life：
Published in Nature：
Transgenic animals – nuclear
transfer：
http://life.nthu.edu.tw/~b851622/Biology/Do
lly%5B1%5D.htm
Name After~ &
Her Unusual Life
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或許大家會問，為什麼桃莉要叫做桃莉呢？有什麼典故
嗎？可別小看這個名字呦，答對了！事實上桃莉這個名
字是很特別的呢！它的特別在於由於桃莉是從乳房細胞
培養出來的，一想到乳房，不禁就讓人聯想到一位非常
有名的女歌星~Dolly Parton，她可是無敵超級大波霸呢
！所以啦，幽默的何威馬博士便將這隻可愛而又具有不
凡身份的小羊ㄇㄟ ㄇㄟ取名叫做桃莉（Dolly）摟。
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小桃莉不平凡的身世在於牠雖然沒有爸爸，可是卻
有三個偉大的媽媽呢！其中一個媽媽名叫Judy，為
白臉芬多斯羊，小桃莉胚胎中的乳房細胞可是從這
個偉大媽媽的胸部來的呦。另一個媽媽名叫Micheal
，牠是黑臉蘇格蘭羊，負責捐贈小桃莉胚胎的卵細
胞。最後一個媽媽則是黑臉蘇格蘭羊，Amy，牠可
是負責懷胎小桃莉的的代理孕母呢！也就是有這三
個偉大的媽，造就了可愛的白臉芬多斯羊ㄇㄟ ㄇㄟ
~桃莉。
Published in Nature
Transgenic Animals – Nnclear Transfer
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進行細胞核移植需仰賴細胞顯
微技術，依次完成下列三個工
作：（1）用紫外光照射受精卵
，破壞原來的細胞核（2）取得
另一個細胞的細胞核（3）再用
極細的玻璃針，重新將新的細
胞核植入卵中。其中破壞卵細
胞核的目的是為了避免當細胞
融合後，不會因為存有多餘的
染色體而影響細胞正常的生長
及分裂。
Dolly the Sheep
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首先，由一隻懷孕的白臉芬多斯母羊中取出乳房細胞，培養
在低養分狀態的條件下一個星期，使細胞停止分裂，進入並
停留在休眠狀態。
準備卵細胞：
從黑臉的蘇格蘭羊中取出卵細胞，然後將卵細胞中央的細胞
核加以破壞。
進行細胞融合：
將去核的卵細胞與乳房細胞放在一起，藉由瞬間電擊的技術
促使細胞融合，另一方面活化細胞內的基因，經過這個步驟
，兩個細胞變會完全融合成單一細胞了，就好比是受精卵一
樣。
培育胚胎：
融合後的胚胎細胞可以藉由培養而不斷分裂，最後由單一細
胞分裂成為一團胚胎細胞。
胚胎細胞的發育：
將培育後的胚胎細胞植入代理孕母黑臉蘇格蘭母羊體內，使
之懷孕，生下來的小羊寶寶，就是今天大家所好奇的白臉桃
莉寶寶呦。
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Cloning Dolly:
http://www.youtube.com/watch?v=CkZV7hl-kXE
http://v.youku.com/v_show/id_XNzU3NTM5NzI=.html
Top 15 Famous Animal Clonings!
http://www.youtube.com/watch?v=qJ9Syd3GqQc&fea
ture=related
Animal technology: http://www.uctv.tv/searchdetails.aspx?showID=14991