Shu-Ping Lin, Ph.D.

Institute of Biomedical Engineering E-mail: splin@dragon.nchu.edu.tw

Website: http://web.nchu.edu.tw/pweb/users/splin/

Genomics: The Technology behind the Human

Genome Project

Electrophoresis 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

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)

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.

Recombinant DNA

Insertion of DNA fragment into an open circular DNA (an empty vector) and thus the formation of cloning vector.

One basic cloning technique begins with the insertion of a foreign gene into a bacterial plasmid (i.e. cloning vector).

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 20.1

Bacterial Plasmids in Gene Cloning

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)

Separation of cell clones containing insert DNA from others

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 20.3

Isolation of cloning vector (bacterial plasmid) & gene-source 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

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.

Making Multiple Copies

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.

This is faster than cloning via recombinant bacteria.

Fig. 20.7

Polymerase Chain Reaction (PCR)

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

In PCR, a three-step cycle--heating, cooling, and replication -- brings about a chain reaction that produces an exponentially growing population of DNA molecules.

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

Restriction fragment length polymorphisms (RFLPs)

Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence

DNA Fingerprinting

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 acrylamide-sequencing gel

DNA Sequencing

Se

qu

en

ce

re

ad

ing

dir

ecti

on

Facts About the Human Genome 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 – 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

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

Dolly the sheep：

Name after~ & her unusual life：

Published in Nature：

Transgenic animals – nuclear transfer：

http://life.nthu.edu.tw/~b851622/Biology/Dolly%5B1%5D.htm

Name After~ & Her Unusual Life

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。

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 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 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。

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&feature=related

Animal technology: http://www.uctv.tv/search-details.aspx?showID=14991