Genetic Engineering. Introduction In the 1970s the field of Biotechnology exploded with the advent of methods producing recombinant DNA 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.


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 can help biologists produce large quantities of a desired protein.

Plasmid

Bacterial cell

Isolateplasmids.

DNA

IsolateDNA.

Cell containingthe gene of interest

Recombinant DNA techniques can be used to produce large quantities of a desired protein and clone genes.

Plasmid

Bacterial cell

Isolateplasmids.

DNA

IsolateDNA.

DNA fragmentsfrom cell

Cut both DNAswith sameenzyme.

Gene ofinterest

Othergenes


Plasmid

Bacterial cell

Isolateplasmids.

Gene of interest

Recombinant DNA plasmids

DNA

IsolateDNA.



Gene ofinterest

Othergenes

Mix the DNAs andjoin them together.


Plasmid

Bacterial cell

Isolateplasmids.

Recombinant bacteria

Gene of interest


Bacteria take up recombinant plasmids.

DNA

IsolateDNA.



Gene ofinterest

Othergenes



Plasmid

Bacterial cell

Isolateplasmids.

Clone the bacteria.

Recombinant bacteriaBacterial clone

Gene of interest



DNA

IsolateDNA.



Gene ofinterest

Othergenes



Plasmid

Bacterial cell

Isolateplasmids.

Find the clone withthe gene of interest.

Clone the bacteria.


Gene of interest



DNA

IsolateDNA.



Gene ofinterest

Othergenes



Plasmid

Bacterial cell

Isolateplasmids.

Some usesof genes

Gene for pestresistance

Gene fortoxic-cleanupbacteria

Genes may beinserted intoother organisms.

Find the clone withthe gene of interest.

The gene and proteinof interest are isolatedfrom the bacteria.

Clone the bacteria.


Gene of interest



Harvestedproteins may beused directly.

Some usesof proteins

Protein for“stone-washing”jeans

DNA


Protein fordissolvingclots

IsolateDNA.



Gene ofinterest

Othergenes


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

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.

Another way to obtain a gene of interest is to: Use reverse transcriptase

and

Synthesize the gene by using an mRNA template


Another approach is to: Use an automated DNA-

synthesizing machine and Synthesize a gene of interest from

scratch


Documents

Genetic Engineering. Introduction In the 1970s the field of Biotechnology exploded with the advent of methods producing recombinant DNA Recombinant DNA