Chapter 16Biotechnology

&Genomics

Gene Cloning: Definition

• Cloning is making an exact replica of something.

• Gene cloning: making many, many copies of identical pieces of DNA.

• Why do we need to clone genes, and how do we do this?

Why Genetic Cloning?Basic Science

To explore a gene’s function, or understand the differences between a mutant and normal gene, researchers need to have a large supply of the gene at hand.

Medicine (Applied Science)We lack the technology to make proteins from scratch, so we need to use bacteria to

make them for us.For bacteria to make a protein, they need to have copies of the genetic information from a gene.

Gene Therapy: Eventually we will be able to replace damaged genes with healthy ones. We can do this already in some cases. To do these kinds of medical miracles, large quantities of the DNA must be made.

Health & the EconomyTransgenic organisms: Organisms that have beneficial DNA transferred to them. We can

modify certain organisms in a beneficial way, or in a way that enhances their value to humans.

How to We Synthesize Cloned DNA?

• There are two basic strategies to clone DNA. Which one we use depends on the source of our original DNA, and what we plan to do with the final results.

• These two strategies are genetic engineering (using recombinant DNA technology) and PCR, or polymerase chain reaction.– We will go through each one of these processes.

Recombinant DNA TechnologyFind a source of DNA

If we are interested in studying a gene’s DNA sequence, and not the protein product, we can use genomic DNA as our source DNA.

If we wish to ultimately look at proteins, we need DNA without introns (right?) This kind of DNA (which we make from the mRNA) is called complimentary DNA, or cDNA (more later)

Put in something you can move it around in – a “Vector”

To actually manipulate the DNA and put it into the host cell, we need a vector. Vectors are simply a “carrier” that allow us to manipulate the DNA sequence that is important to us.

Vectors can be either plasmids or viruses.

Note: DNA from two or more different sources (bacteria or virus DNA, and another organism’s DNA such as humans) is called Recombinant DNA.

Genomic DNA

mRNA

Reverse transcriptasecDNA

Plasmid

virus

Recombinant DNA TechnologyEnzymes as Tools

To actually put a foreign piece of DNA into a plasmid or vector, we literally cut and paste DNA.

We can cut it VERY specifically, by taking advantage of the primitive bacterial immune system.

This consists of a variety of enzymes that cut DNA at specific sequences – we call these restriction enzymes.

Normally, bacteria use these to destroy foreign DNA (such as that coming in from bacteriaphages).

Humans have purified hundreds of these enzymes, creating a library of precise tools that can cut DNA at almost any sequence!

Once we cut the DNA, we can paste in our cDNA (or genomic DNA). To do this, another enzyme, DNA ligase, is used.

Plasmid

Restriction enzyme

cDNA insert

DNA Ligase

Recombinant DNA Technology

The final result is a recombinant plasmid with useful DNA in it.

Host

once we have our vector, we need a way to actually use it.

We stick it special strains of bacteria such as e. coli

Transformation

You should already be familiar with this concept.

PCR: Polymerase Chain Reaction

• Another way to clone DNA – in a test tube – is to allow it to undergo many rounds of replication.

• The basic idea behind PCR is simple: DNA is replicated in a test tube over and over again.

Double-stranded DNAPCR

CGTATCGA

AGGTCCTG

DNA is cooled – a little bit.In order to begin replication in a test tube, DNA polymerase needs a “landing pad” that consists of double-stranded DNA to extend.We use small pieces of DNA, called “primers”, to set the starting points of replication.Once primers bind (called hybridization), DNA polymerase extends each strand.

5’ 3’

3’ 5’

Increased heat “melts” the DNA

double helix.

5’ 3’

3’ 5’

DNA is “melted” (the hydrogen bonds between base pairs break because of heat)

elongation

elongation

Double-stranded DNAPCR

Each new cycle produced twice the amount of DNA from the previous cycle. There is a geometric expansion.Within a few hours, a single piece of DNA can be cloned into over a million pieces.

The cycle is repeated.248

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1048576

Sources of DNA

• Primers can be easily synthesized in a lab.• So if we know at least a little bit of a gene’s

sequence, we can clone it.• There are thousands of unexplored genes in

the human genome, waiting to be investigated!

Quick example

• A researcher finds a correlation between a kind of leukemia and a strange looking karyotype.

• “8 out of 10 patients with this form of leukemia also have a translocation between chromosome 15 and 17.”

• We look at the genes in this region. We find a few that look “interesting” (maybe there’s an unknown gene that has sequence similarity with a yeast gene known to be involved in cell growth)

Quick example

• The researcher makes a primers that flank the gene of interest.

• They do PCR on genomic DNA from people that don’t have leukemia and people that do.– They isolate a “wild type” and “mutant” gene.

• They put the gene into a plasmid, and clone it in bacteria

• They sequence both genes, and find that there is a mutation.

Quick example

• They go back to the patient and “wild type” person, and purify the mRNA from their cells

• They use reverse transcriptase to make cDNA• They use PCR to make a LOT of this cDNA• They use genetic engineering to put this piece

of cDNA into a plasmid.• They transform bacteria• The bacteria produce wild-type or normal

protein.

Quick example

• The researcher can purify the 5two kinds of protein, and perform various experiments on it.

• THE END

• PCR can also be used to determine whether individuals have certain disease genes.

• Genetic screening can show if people are predisposed to certain diseases, and can therefore allow them to alter their lifestyle to encourage an improved quality and longevity.

DNA Fingerprinting

• Take someone’s DNA and cut it with restriction enzymes.• Because there are SO many sequences, there will be – due to

chance alone – many regions of this person’s genome that is cut by the enzyme.

• This DNA is run on a gel, and a unique set of bands can be detected. This is called a person’s “DNA fingerprint” or “genomic fingerprint”

• We use this for many reasons:– Identifying the father– Criminal cases when “DNA evidence” is used– Identifying distinct populations of organisms– Many other uses – limited only by human imagination.

Uses of cloning

• Biotechnology is a huge business.– Genentech has a $72 B market cap– Amgen has a 56.7 B market cap– Compare: GE has a 378 B cap; Apple is 170 B

Uses of cloning

• Transgenic Organisms– Organisms that contain DNA that was not part of the

original genome.• Transgenic bacteria

– Produce important proteins and compounds such as insulin (diabetes), and phenylalanine (for nutrasweet)

– They can use oil as a source of food.– Produce complex organic compounds we can’t easily

synthesize (phenylalane for nutrasweet)• Transgenic plants & animals

– Interesting list here

In the News• The end of the stem cell debate?

– The science news article• Once the kinks are worked out, "the whole field is going to

completely change," says stem cell researcher Jose Cibelli of Michigan State University in East Lansing. "People working on ethics will have to find something new to worry about.“

• Fox News Article (by… Father John)– Shows that the good scientists really don’t have an ego.

• Professor Wilmut will not take advantage of his licence from the United Kingdom to attempt human embryonic cloning because he is now convinced that two independent research teams in Japan and the United States have discovered a process that is much more efficient than therapeutic cloning (his own discovery) and offers a more realistic and timely hope for therapies of serious illnesses, such as stroke, heart disease and Parkinson’s disease.

• 1. genetically engineered microorganisms (GEMS) • 2. transgenic plants • 3. transgenic animals • D. Gene therapy • 1. Discuss the concept and importance. • 2. List technical approach(es) such as viral vectors. • 3. Provide examples of the therapy such as cystic fibrosis,

adenosine deaminase deficiency. • 4. Define somatic cell and germ cell gene therapy. • 5. List positive and negative aspects with a discussion of

eugenics.

Genomics

• Genome Sequences• Proteomics• Bioinformatics