Gene Cloning and DNA Analysis: An introduction

T. A. Brown. 6th edition 2010 Published by Blackwell Science Ltd

& 140.128.147.174/yclclass/ =>2011

Part I The Basic Principles of Gene Cloning and DNA Analysis 1 Why gene cloning and DNA analysis are important 2 Vectors for gene cloning: plasmids and bacteriophages 3 Purification of DNA from living cells 4 Manipulation of purified DNA 5 Introduction of DNA into living cells 6 Cloning vectors for E. coli 7 Cloning vectors for eukaryotes 8 How to obtain a clone of a specific gene 10 The polymerase chain reaction Part II The Applications of Gene Cloning and DNA Analysis in Research 11 Sequencing genes and genomes 12 Studying gene expression and function 13 Studying genomes Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 14 Production of protein from cloned genes 15 Gene cloning and DNA analysis in medicine 16 Gene cloning and DNA analysis in agriculture 17 Gene cloning and DNA analysis in forensic medicine

1 Why Gene Cloning and DNA Analysis are Important

1.1 The early development of genetics 1.2 The advent of gene cloning and the polymerase chain reaction 1.3 What is gene cloning? 1.4 What is PCR? 1.5 Why gene cloning and PCR are so important
1.5.1 Obtaining a pure sample of a gene by cloning 1.5.2 PCR can also be used to purify a gene

1.6 How to find your way through this book

Middle of the 19th century: Gregor Mendel

A set of rules to explain inheritance of biological characteristics Control by a factor (gene)

1900: rediscovery of Mendels laws make the birth of genetics

The early development of genetics

1903: W. Sutton
genes reside on chromosomes

1910: T. H. Morgan
developed techniques for gene mapping By 1922, analysis of relative positions of >2000 genes on 4 chromosomes of Drosophila melanogaster

DNA is the genetic material

1944: Avery, Macleod, & McCarty 1952: Hershey & Chase

The early development of genetics

Discovery of role of DNA
A tremendous stimulus to genetic research Many famous biologists contributed to the second great age of genetics Delbrck, Chargaff, Crick, & Monod

1952 - 1966
structure of DNA was elucidated genetic code cracked processes of transcription & translation described

The advent of gene cloning and the polymerase chain reaction

1971-1973, a whole new methodology was developed
recombinant DNA technology or genetic engineering at their core process of gene cloning sparked another great age of genetics

1990s: led to rapid & efficient DNA sequencing techniques

enables structure of individual genes to be determined

A culmination () with massive genome sequencing projects (2000) Human project was completed in 2000

The advent of gene cloning and the polymerase chain reaction

Procedures for studying regulation of individual genes
To understand how aberrations in gene activity can result in human diseases such as cancer

Modern biotechnology
Put genes to work in production of proteins & other compounds needed in medicine & industrial processes

1980s
Excitement engendered by gene cloning revolution was at its height

The advent of gene cloning and the polymerase chain reaction

1985: Kary Mullis
The polymerase chain reaction (PCR) during a drive along coast of California one evening His brainwave was an exquisitely simple technique that acts as a perfect complement to gene cloning PCR has made easier many of the technique Dancing naked in the that were possible but difficult to carry out when gene cloning was used on its ownmind eld

The advent of gene cloning and the polymerase chain reaction

DNA analysis traditional range
Medicine, agriculture & biotechnology PCR has extended its range & enabled MB to find new applications

New disciplines ()
Archaeogenetics (), molecular ecology (), DNA forensics Human evolution, impact of environmental 40change on biosphere, fight against crime yrs passed, we are still riding rollercoaster &there is
no end to excitement in sight.

What is gene cloning?

1. A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule 2. The vector transports the gene into a host cell
which is usually a bacterium, although other types of living cell can be used

3. Within the host cell the vector multiplies

producing numerous identical copies not only of itself but also of the gene that it carries

What is gene cloning?

Transformation 42 C, 90
clone (/ )

What is gene cloning?

4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny () & further vector replication takes place 5. After a large number of cell divisions, a colony, or clone, of identical host cells is produced
Each cell in the clone contains one or more copies progeny () of the recombinant DNA molecule The gene carried by the recombinant molecule is now said to be cloned

What is gene cloning?

What is PCR?
1.The mixture is heated to 94 C
The hydrogen bonds are broken, causing the molecules to denature

2.The mixture is cooled down to 50-60 C

Two strand of each molecule could join back together, but most do not Because the mixture contains a large excess of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA molecules at specific positions

What is PCR?

What is PCR?
3. The temperature is raised to 74 C
Mixture: Taq DNA polymerase, dNTP It attaches to one end of each primer & synthesizes new strands of DNA, complementary to template DNA 2 => 4 strands

4. Temperature is increased back to 94 C

Denature again A second cycle of denaturation-annealing-synthesis (4 => 8 strands) Repeat 30 cycles: >130 million (106) new ds molecules

Why gene cloning and PCR are so important?

Both techniques can provide a pure sample of an individual gene, separated from all the other gene in the cell

Obtaining a pure sample of a gene by cloning

DNA fragment to be cloned is one member of a mixture of many different fragments
This mixture could indeed be the entire genetic complement of an organism

Each fragments becomes inserted into a different vector to produce a family of recombinant DNA molecule
One of which carries the gene of interest Only one recombinant DNA molecules

is

transported into any single host cell

The gene is now separated away from all the other gene in the original mixture

Figure 1.3 Cloning allows individual cloningABC A B fragments of DNAamplify to be purifiedC

A C B

ABC

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Figure 1.4 The problem of selection

>4000 different genes in genome of E. coli 5X as many genes in human genome

How to separated from all the other gene

Success or failure of a gene cloning
Ability to identify the particular clone of interest from the many different ones that are obtained

Selection
Chap 8: a variety of different strategies can be used

Once a gene has been cloned there is almost no limit to the information that can be obtained about it structure & expression
Methods for studying the structure & expression of a cloned gene are described in chap 10 & 11

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PCR can also be used to purify a gene

Figure 1.5 Gene isolation by PCR Outcome is the same as with a gene cloning Automatically selected A rapid technique
Complete in a few hours Cloning: months

Much less complicated Tremendous sensitivity

PCR has two limitations

The sequences of these annealing sites must be known
PCR cannot be used to isolate genes that have not been studied before That has to be done by cloning

The length of DNA sequence that can be copied by PCR

5 kb: copy fairly easily < 40 kb: using specialized techniques > 40 kb: Many genes of human & other vertebrates Cloning is the only way Gene cloningmust be used of isolating long genes or those

that have never been studied before.

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Clinical applications of PCR

Even if the sequence of a gene is not known
A gene that has been isolated & sequenced from mouse could therefore be used to design a pair of primers for isolation of the equivalent gene from humans

PCR => sequenced

A PCR of human globin genes is used to test for the presence of mutations Thalassaemia, sickle cell anemia

Detect viruses at earliest stages of an infection

Use of primers specific for the DNA of a diseasecausing virus Even if there is just one DNA molecule in starting mixture

How to find you way through this book

Part I: we deal with the basic principle
Chap2-9 Techniques for handling DNA

Part II: how genes and genome are studied Part III: broader applications
Biotechnology, medicine, agriculture & forensic science

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10%

45%
Chapter 1-8

45%
Chapter 9-16

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