Download Recombinant DNA

Genetic Engineering
Introduction
 In the 1970s the field of Biotechnology exploded with
the advent of methods producing recombinant DNA
 Recombinant DNA is formed when scientists
combine pieces of DNA from two different sources
 Recombinant DNA technology is now widely used in
genetic engineering (the manipulation of genes
for practical purposes)
Applications
 Genetic Engineering has allowed us to…

Mass produce insulin and many other important human
proteins using bacteria, yeasts, and mammalian cells

Produce many vaccines against infectious diseases

Improve productivity & nutritional value of agriculturally
important plants
Genetic Engineering Basics
 DNA is the “molecular” language that is common to
all life.
 All living organisms use DNA to store their genetic
information and direct protein synthesis. And
because of this, organisms are capable of expressing
genes unique to any other organisms or species
Genetic Engineering Basics
 Genetic engineering in practice is accomplished by…
1.
Isolating/obtaining a gene of interest
2.
Producing recombinant DNA (by inserting the gene of
interest into another DNA molecule)
3.
Inserting the recombinant DNA into the host organism
Recombinant DNA Techniques


Bacteria are the workhorses of modern biotechnology.
To work with genes in the lab, biologists often use bacterial
plasmids, small, circular DNA molecules that are separate
from the much larger bacterial chromosome.
Recombinant DNA Techniques

Plasmids:

Easily incorporate foreign DNA

Are readily taken up by bacterial cells

Can act as vectors (DNA carriers that move genes from one cell to
another)

Are ideal for gene cloning (producing multiple identical copies of
a gene-carrying piece of DNA)
Recombinant DNA Techniques
Recombinant DNA techniques can help
biologists produce large quantities of a
desired protein.
Isolate
DNA.
Bacterial cell
Isolate
plasmids.
Cell containing
the gene of interest
Plasmid
DNA
Recombinant DNA techniques can be used to
produce large quantities of a desired protein and
clone genes.
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Bacterial cell
Isolate
plasmids.
DNA fragments
from cell
Isolate
DNA.
Cell containing
the gene of interest
Plasmid
DNA
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Plasmid
DNA
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Recombinant bacteria
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Bacterial clone
Recombinant bacteria
Clone the bacteria.
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Bacterial clone
Recombinant bacteria
Clone the bacteria.
Find the clone with
the gene of interest.
Cut both DNAs
with same
enzyme.
Gene of Other
interest genes
Gene of interest
Bacterial cell
DNA fragments
from cell
Isolate
DNA.
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Isolate
plasmids.
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Plasmid
DNA
Bacterial clone
Recombinant bacteria
Clone the bacteria.
Find the clone with
the gene of interest.
Some uses
of genes
Gene for pest
resistance
Some uses
of proteins
Protein for
dissolving
clots
Gene for
toxic-cleanup
bacteria
Genes may be
inserted into
other organisms.
The gene and protein
of interest are isolated Harvested
proteins may be
from the bacteria.
used directly.
Protein for
“stone-washing”
jeans
Cutting and Pasting DNA via Restriction Enzymes

Recombinant DNA is produced by combining two ingredients:
A bacterial plasmid
 The gene of interest


To combine these ingredients, a piece of DNA must be spliced
into a plasmid.
© 2010 Pearson Education, Inc.
Cutting and Pasting DNA via Restriction Enzymes


This splicing process can be accomplished by:

Using restriction enzymes, which cut DNA at specific
nucleotide sequences and

Producing pieces of DNA called restriction fragments with
“sticky ends” important for joining DNA from different sources
DNA ligase connects the DNA pieces into continuous strands
by forming bonds between adjacent nucleotides.
Cutting &
Pasting DNA
Obtaining the Gene of Interest

How can a researcher obtain DNA that encodes a particular
gene of interest?

A “shotgun” approach yields millions of recombinant plasmids
carrying many different segments of foreign DNA.

A collection of cloned DNA fragments that includes an organism’s
entire genome (a complete set of its genes) is called a genomic
library.
Obtaining a gene of interest
 Methods for detecting a gene of interest depend on
the nucleotide sequence of the gene.
 When at least part of the nucleotide sequence of a
gene is known, scientists can use nucleic acid probes
to find the gene
Nucleic Acid Probes
 A nucleic acid probe is a short sequence of
nucleotides that is complimentary to the sequence of
the gene of interest. The probe is also labeled with a
radioactive isotope or a fluorescent dye.
Obtaining a gene of interest

Another way to obtain a gene
of interest is to:

Use reverse transcriptase and

Synthesize the gene by using
an mRNA template
Obtaining a gene of interest

Another approach is to:
Use an automated DNA-synthesizing
machine and
 Synthesize a gene of interest from
scratch
