Under the Guidance of, Prepared By, Mrs.K.Geetha., Raviteja.A

Assistant professor Reg.No.10T21S0101

CMR COLLEGEOF PHARMACY KANDLAKOYA (V), HYDERABAD - 501401

CONTENTS

1. INTRODUCTION2. DNA CLONING3. STEPS IN DNACLONING4. POLYMERASE CHAIN REACTION5. EXPRESSION6. LIBRARY CONSTRUCTION7. APPLICATIONS OF DNA CLONING

Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism.

DNA, or deoxyribonucleic acid, is the hereditary material,it is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is Called as mtDNA).

INTRODUCTION

•DNA is made up of four nucleotides: A, C, G, and T. A always pairs with T.

C always pairs with G.

The two strands of DNA are in an antiparallel configuration.

Two complementary DNA strands will separate when heated, and will spontaneously pair together again (hybridize) when cooled.

STEPS IN DNA CLONING

• The following steps in the cloning process:• 1 Isolation of vector and gene-source DNA

• 2. Insertion of DNA into the vector.

• 3 Introduction of the cloning vector into cells.

How do we monitor each of these steps?

Cloning-:The production of identical copies of molecules, cells, or replicating nucleic acid sequences from organismsfrom a single ancestor. The DNA of interest can then be propagated in a foreign host cell (bacterial plasmid) This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.

• A cloning vector is a plasmid that can be modified to carry new genes.

• Plasmids useful as cloning vectors must have:– An origin of replication.– A selectable marker (antibiotic resistance gene, such

as ampr and tetr).– Multiple cloning site (MCS) (site where insertion of

foreign DNA will not disrupt replication or inactivate essential markers).

– Easy to purify away from host DNA.– There are also cosmid vectors, bacterial

artificial chromosomes, and yeast artificial chromosomes.

• After culture growth, the clone fragment can be recovered easily. The cells are lysed and the DNA is isolated and purified.

• A DNA fragment can be kept indefinitely if mixed with glycerol in a –70 degrees C freezer

Tetr

Ampr

OripBR322

4361bp

OripUC18

Ampr

MCSLacZ

Older cloning vector

Newer cloning vector

Plasmids – vehicles for cloning

Restriction Enzymes: These Enzymes cuts the DNA from any organism at

specific sequences of a few nucleotides, generating a reproducible set of fragments.

Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences

DNA Ligases: insert DNA restriction fragments into replicating DNA

molecules producing recombinant DNA.

Restriction enzymes nomenclature

• EcoRI – Escherichia coli strain R• BamHI – Bacillus amyloliquefaciens • DpnI – Diplococcus pneumoniae, • HindIII – Haemophilus influenzae, • BglII – Bacillus globigii,• PstI – Providencia stuartii 164,• Sau3AI – Staphylococcus aureus 3A,• KpnI – Klebsiella pneumoniae, 1st

DNA cloning requires restriction endonuclease and DNA ligase

Consider a plasmid with a unique EcoRI site:

5' NNNNGAATTCNNNN 3' 3’ NNNNCTTAAGNNNN 5'

An EcoRI restriction fragment of foreign DNA can be inserted into a plasmid having an EcoRI cloning site by: a) cutting the plasmid at this site with EcoRI, b) annealing the linearized plasmid with the EcoRI foreign DNA fragment, and,c) sealing the nicks with DNA ligase.

5' NNNNGAATTCNNNN 3' 3' NNNNCTTAAGNNNN 5’

This results in a recombinant DNA molecule.

Plasmid vector

+

DNA fragment to be cloned

Enzymatically insert DNA into plasmid vector

Recombinant plasmid

Mix E.coli cells with plasmids in presence of CaCl2 Culture on

nutrient agar plates containing ampicillin

Cells that do not take up plasmids die on ampicillin plates

Transformed E.coli cell survives

Bacterial chromosome

Independent plasmid replication

Cell multiplication

Amp R.

Amp R.ORI

• One basic cloning technique begins with the insertion of a foreign gene into a bacterial plasmid.

A thermophilic (heat-loving) bacteria called Thermus aquaticus is the source of Taq DNA polymerase used in PCR reactions.

Polymerase Chain Reaction (PCR)A method for amplifying DNA segments using cycles of denaturation, annealing to primers,and DNA polymerase-directed DNA synthesis

A typical PCR protocol

Key features of DNA replication are used in DNA sequencing

• DNA synthesis occurs in the 5´ to 3´ direction.

• DNA synthesis requires a template and a primer.

• DNA replication is semi-conservative (one strand copied).

• DNA replication is carried out by an enzyme called DNA polymerase.

Expression

The main function of an expression vector is to yield the product of a gene, therefore a strong promoter is necessary. The more mRNA is produced, the more protein product is made.

Vectors that can yield the protein products of the cloned genes.

Two elements that are required for active gene expression: a strong promoter and a ribosome binding site.

Expression

Why?You want the cloned gene to make its product,

normally a protein. Identifying gene from library requires

expression.To overproduce the protein and purify it.For in vivo studies of the protein.

Library ConstructionA library is a collection of different cloned DNAs from a single

source that are present in different copies of a particular cloning vector.

To generate a library with a million clones for example, you need to recover a million independent colonies carrying plasmids or a million independent phage plaques. By pooling together all the independent clones you get the library

Though simple in principle, libraries are difficult to make well

• Applications The various applications of gene cloning include recombinant protein production, genetically modified organism, DNA fingerprinting, diagnostic kits and gene therapy.

• Use of RDNA Technology to produce human Insulin

• Recombinant protein By inserting the gene for a rare protein into a plasmid and expressing it in bacteria, large amounts of recombinant protein can be produced.

Genetically modified organisms Introducing a foreign gene into an organism which can propagate creates a genetically modified organism. Transgenic sheep have been created.

DNA fingerprinting Hybridizing of genomic DNA with probes that recognize simple nucleotide repeats gives a pattern that is unique to an individual and can be used as a fingerprint. This has applications in forensic science, animal and plant breeding and evolutionary studies.

Medical diagnosis The sequence information derived from cloning medically important genes has allowed the design of many diagnostic test kits which can help predict and confirm a wide range of disorders.

Gene therapy Attempts to correct a genetic disorder by delivering a gene to patient are described as gene therapy.

Animals Can Be Cloned by Several Methods

• Embryo splitting– After in vitro fertilization, early embryonic cells are

divided and grown into clones

• Nuclear transfer (cell fusion)– Enucleated eggs are fused with embryonic or

adult cells and grown into clones– Dolly the sheep

Plants Can Be Cloned from Single Cells

• 1950s: Charles Steward grew individual carrot cells in the laboratory by using special nutrients

• Single cells grew and divided to form a ball of undifferentiated cells (callus)

• Calluses transferred to a different medium grew into full-size carrots (clones)

• Development of methods for cloning higher plants and animals represents a significant advance in genetic technology– Improving crops– Producing domestic animals

Technology now exists to sequence everyone’s DNA

Took just 4 months,$1.5 million to obtain the entire DNA sequence of James Watson.

Understanding the arrangement of genes may help understand disease

Genome resources for many organisms are available

Why is DNA Cloning Important?

• DNA clones are used to find genes, map them, and transfer them between species

• Cloning technology is used to find carriers of genetic disorders, perform gene therapy, and create disease-resistant plants

Because of recent technological advancements cloning in the animals and humans has been an issue and is strictly banned, Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation has been common practice in the horticultural world for hundreds of years.

Thus Cloning manipulate a novel gene in many different ways with the goal of uncovering its unique role in the cell.