General GeneticsDr. Attya Bhatti

Genetic Engineering

Also known asGene manipulationGenetic modifications recombinant DNA technology,New Genetics

Means: altering the genes in a living organism to produce a

Genetically Modified Organism (GMO) with a new genotype.

Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism altering an existing gene so that its product is changed Changing gene expression so that it is translated more

often or not at all.

History of Genetics Since 1900.

19901910192019301940195019601970198019902000

Genetic MappingTransformation demonstration

Microbial Genetics

Mendelian Genetics

Gene Manipulation

Development of techniquesApplications

Molecular Genetics

Basic Concepts of Genetic Engineering

Recombinant DNA technology is a set of methods used to

locate, analyze, alter, study, and recombine DNA sequences.

It is used to probe the structure and function of genes,

address questions in many areas of biology, create

commercial products, and diagnose and treat diseases.

Steps in Genetic Engineering

Isolate the gene

Insert it in a host using a vector

Produce as many copies of the host as possible

Separate and purify the product of the gene

Generation of DNA Fragments

Joining to a vector or carrier Molecule

Introduction into a host cell for amplification

Selection of required sequence.

Step 1: Isolating the Gene

Step 1: Alternative Method (using reverse

transcriptase)

Reverse transcriptase

mRNA converted into cDNA

Complementary strand produced using DNA polymerase

Advantage – more mRNA in cell than DNA

Step 2: Inserting Gene into Vector

Vector – molecule of

DNA which is used to

carry a foreign gene

into a host cell

Step 3: Inserting Vector into Host

Replica Plating

Step 4: Multiplication of the Host Cells by

Cloning

Large scale fermenters by cloning

All genetically identical because of asexual

reproduction

Step 5: Extraction of desired gene

product.

Genetically engineered corn, which produces a toxin that kills insect pests, now comprises over 30% of all corn grown in the United States.

Recombinant DNA technology has been used to create genetically modified crops.

Working at the Molecular Level

Recombinant DNA technology requires special methods

because:

Individual genes make up a tiny fraction of the cellular

DNA and they cannot be seen.

Recombinant DNA Techniques

Methods for locating specific DNA sequences:

Techniques for cutting DNA at precise locations

Procedures for amplifying a particular DNA sequence billions

of times, producing enough copies of a DNA sequence to

carry out further manipulations

Methods for mutating and joining DNA fragments to produce

desired sequences

Procedures for transferring DNA sequences into recipient

cells

Restriction Enzymes Also called restriction endonucleases that recognize and make

double-stranded cuts in the sugar–phosphate backbone of DNA

molecules at specific nucleotide sequences.

These enzymes are produced naturally by bacteria, where they

are used in defense against viruses.

In bacteria, restriction enzymes recognize particular sequences

in viral DNA and then cut it up. A bacterium protects its own

DNA from a restriction enzyme by modifying the recognition

sequence, usually by adding methyl groups to its DNA.

Types of Restriction Enzymes

Three types of restriction enzymes have been isolated from

bacteria

Type I restriction enzymes

Type II restriction enzymes

Type III restriction enzymes

Type I Restriction Enzymes

Recognize specific sequences in the DNA

Cut the DNA at random sites that may be some distance

(1000 bp or more) from the recognition sequence

Type II Restriction Enzymes Recognize specific sequences

Cut the DNA within the recognition sequence

Virtually all work on recombinant DNA is done with type II restriction enzymes

Type III Restriction Enzymes Recognize specific sequences

Cut the DNA at nearby sites

Usually about 25 bp away

The number of restriction sites is related to the number of fragments produced when DNA is cut by a restriction enzyme

VectorsIs a vehicle for delivering genetic material

such as DNA to a cell

Cloning vectors

Plasmid vectors

Bacteriophage vectors

Cloning Vectors

is a stable, replicating DNA molecule to which a foreign DNA

fragment can be attached for introduction into a cell.

Three important characteristics:

an origin of replication which ensures that the vector is

replicated within the cell.

Selectable markers, which enable any cells containing

the vector to be selected or identified.

one or more unique restriction sites into which a DNA

fragment can be inserted.

Three characteristics of an idealized cloning vector

Plasmid vectors

Plasmids are circular DNA molecules that exist naturally in

bacteria

contain origins of replication and are therefore able to

replicate independently of the bacterial chromosome

Used in cloning have been constructed from the larger,

naturally occurring bacterial plasmids

Plasmid vectors

Example:

pUC19 plasmid

has an origin of replication

two selectable markers—an ampicillin-resistance

gene and a typical cloning vector

The pUC19 plasmid is a typical cloning vector

Bacteriophage Vectors

Bacteriophages offer a number of advantages as cloning

vectors.

Most widely used bacteriophage vector is bacteriophage ,

which infects E. coli

Advantages:

High efficiency with which it transfers DNA into bacteria

cells

Viewing DNA Fragments

DNA fragments can be separated, and their sizes can be

determined with the use of gel electrophoresis.

The fragments can be viewed by

Using a dye that is specific for nucleic acids

By labeling the fragments with a radioactive or chemical

tag.

Gel electrophoresis can be used toseparate DNA molecules on the basis of their size and electrical charge

Cloning Genes

Identical copies (clones) of the original piece of DNA are

produced

DNA fragments can be inserted into cloning vectors, stable

pieces of DNA that will replicate within a cell.

Cloning vectors must have an origin of replication, one or

more unique restriction sites, and selectable markers.

Plasmids are commonly used as cloning vectors.

Applications

Basic Research on gene Structure and Function

Production of useful proteins by novel methods

Generation of transgenic plants and animals

Medical diagnostics and treatment

Applications

In addition to providing valuable new information about the

nature and function of genes, recombinant DNA technology

has many practical applications

Include the production of pharmaceuticals and other

chemicals, specialized bacteria, agriculturally important

plants, and genetically engineered farm animals

ApplicationsOligonucleotide Drugs:

Oligonucleotide drugs are short pieces of DNA or RNA

that prevent the expression of particular genes.

Genetic Testing:

The identification and cloning of many important disease

causing human genes has allowed the development of

probes for detecting disease-causing mutations.

Applications Genetic Testing:

The identification and cloning of many important disease

causing human genes has allowed the development of

probes for detecting disease-causing mutations.

Gene Therapy:

Ultimate application of recombinant DNA technology

is gene therapy the direct transfer of genes into humans

to treat disease

Gene Mapping:

Significant contribution of recombinant DNA technology has

been to provide numerous genetic markers that can be used in

gene mapping.

One group of markers used in gene mapping comprises

restriction fragment length polymorphisms (RFLPs, pronounced

rifflips).

Applications

Applications DNA Fingerprinting:

Restriction fragment length polymorphisms are often found in

non coding regions of DNA and are therefore frequently quite

variable in humans.