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.