Biotechnology

Dolly and surrogate Mom

Genetically modified rice.

Biotechnology

Biotechnology, defined broadly, is the engineering of organisms for useful purposes.

Often, biotechnology involves the creation of hybrid genes and their introduction into organisms in which some or all of the gene is not normally present.

Fourteen month-old genetically engineered (“biotech”) salmon (left) and standard salmon (right).

Biotechnology

We’ll examine:

Gene cloning for pharmaceutical production

DNA fingerprinting

Recombinant DNA, Gene Cloning, and Pharmaceutical Production

DNA can be cut at specific sequences using restriction enzymes.

This creates DNA fragments useful for gene cloning.

These are mature and widely utilized biotechnologies.

Restriction Enzymes are Enzymes That Cut DNA Only at Particular Sequences

The enzyme EcoRI cutting DNA at its recognition sequence

Different restriction enzymes have different recognition sequences.

This makes it possible to create a wide variety of different gene fragments.

Restriction enzyme animation

DNAs Cut by a Restriction Enzyme Can be Joined Together in New Ways

These are recombinant DNAs and they often are made of DNAs from different organisms.

Plasmids are Used to Replicate a Recombinant DNA

Plasmids are small circles of DNA found in bacteria.

Plasmids replicate independently of the bacterial chromosome.

Replication often produces 50-100 copies of a recombinant plasmid in each cell.

Pieces of foreign DNA can be added within a plasmid to create a recombinant plasmid.

Harnessing the Power of Recombinant DNA Technology – Human Insulin Production by Bacteria

Human Insulin Production by Bacteria

6) join the plasmid and human fragment

and cut with a restriction enzyme

Human Insulin Production by Bacteria

Mix the recombinant plasmid with bacteria.

Screening bacterial cells to learn which contain the human insulin gene is the hard part.

Route to the Production by Bacteria of Human Insulin

A fermentor used to grow recombinant bacteria.

This is the step when gene cloning takes place.

The single recombinant plasmid replicates within a cell.

Then the single cell with many recombinant plasmids produces trillions of like cells with recombinant plasmid – and the human insulin gene.

One cell with the recombinant plasmid

Route to the Production by Bacteria of Human Insulin

The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene.

Route to the Production by Bacteria of Human Insulin

Overview of gene cloning. Cloning animati

on

Reverse transcription: cDNA Cloning

Suppose we need to clone a gene containing lots of introns.  What will happen when the bacterium tries to express it?

To overcome this problem, we can start with mRNA isolated  from tissues that produce the desired protein.  We then use reverse transcriptase enzyme (produced by a retrovirus related to HIV) to reverse transcribe the mRNA into a DNA molecule that now is free of introns.  Now we can ligate "sticky ends" onto the cDNA and recombine it into a phage or plasmid vector.

Or: Use Proteins sequence - BLAST website to get DNA sequence and PCR to make multiple copies of cDNA that can then be cloned.

DNA, the Law, and Many Other Applications – The Technology of DNA Fingerprinting

A DNA fingerprint used in a murder case.

What are we looking at? How was it produced?

The defendant stated that the blood on his clothing was his.

DNA Fingerprinting Basics

Different individuals carry different alleles.

Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain.

DNA Fingerprinting Basics

If DNA is cut with a restriction enzyme that recognizes sites on either side of the region that varies, DNA fragments of different sizes will be produced.

A DNA fingerprint is made by analyzing the sizes of DNA fragments produced from a number of different sites in the genome that vary in length.

The more common the length variation at a particular site and the greater the number the sites analyzed, the more informative the fingerprint.

A Site With Three Alleles Useful for DNA Fingerprinting

DNA fragments of different size will be produced by a restriction enzyme that cuts at the points shown by the arrows.

The DNA Fragments Are Separated on the Basis of Size

The technique is gel electrophoresis.

The pattern of DNA bands is compared between each sample loaded on the gel.

Gel electrophoresis animation

Possible Patterns for a Single “Gene” With Three Alleles

In a standard DNA fingerprint, about a dozen sites are analyzed, with each site having many possible alleles.

A DNA Fingerprint

When many genes are analyzed, each with many different alleles, the chance that two patterns match by coincidence is vanishingly small.

DNA detective animation

HGP fingerprinting page

Cloning animationCloning animationCloning animationCloning animation

Cloning animation

DNA and the Law

SLT 3/8/05

Some applications of DNA fingerprinting in the justice system.

Genetically Modified Foods

Many of our crops in the US are genetically modified.

Should they be?

GM Crops are Here Today

Source: Pew Initiative on Food and Biotechnology, August 2004.

Methods for Plant Genetic Engineering are Well-Developed and Similar to Those for Animals

Golden Rice is Modified to be Provide a Dietary Source of Vitamin A

Worldwide, 7% of children suffer vitamin A deficiency, many of them living in regions in which rice is a staple of the diet.

Golden rice (yellow) with standard rice (white).

Genetically Modified Crops

Genetically Modified Cotton (contains a bacterial gene for pest resistance)

Standard Cotton

Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops

The jury’s still out on the magnitude of GM crop’s ecological impact, but the question is debated seriously.

Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops