4.4 Biotechnological Tools and Techniques

Human Genome Project A commitment by the scientific community

to determine the location and structure of all genes in the human chromosomes

Scientists sequence the genes of a section of a chromosome and pool the information together.

Having a map of the sequence of nucleotides of human DNA can lead to mapping of genes (listing and finding the locus of each human gene)

Human Genome Project: OUTCOME Improved understanding of genetic diseases

Production of medicines (based on DNA sequences) to cure diseases

Genetic screening (and preventative medicine)

Focused research

Provides more information about the evolutionary paths between species

Recombinant DNA: a fragments of DNA composed of sequences originating from at least 2 different sources

Why? – ex: Agricultural Benefits: increase in crop yields; disease resistance; crop longevity

Genetic Engineering The deliberate manipulation of genetic

material

It is possible to move genetic material from one speicies to another because the genetic code is universal

◦ All organisms use the same nitrogenous bases◦ In all organisms, the same codon codes for the

same amino acid (ie: in all species, AUG codes for the amino acid methionine)

Genetic Engineering Hypothetically….. If we could insert a gene into another

organism’s genome (DNA), that organism would express that gene (make the protein the gene codes for)

To do this, we would need “molecular scissors” to cut the gene sequence from our original source and “molecular glue” to insert the gene sequence into the host organism’s DNA

Also known as Restriction Enzymes are “molecular scissors” that can cleave (cut) double stranded DNA at a specific base-pair sequences.

Bacterial enzymes

Recognition Site: the specific sequence where the restriction enzyme makes its cut.◦ Usually palindromic◦ ~4 to 8 nucleotides long

STICKY ENDS

STICKY ENDS: both fragments of the newly cleaved DNA have DNA nucleotides lacking complimentary bases.

BLUNT ENDS: The ends of the DNA fragments are fully paired.

Restriction enzymes that produce sticky ends are more useful because these DNA fragments can easily be joined to other DNA sticky ends fragments made by the same restriction enzyme.

Can easily be used to create recombinant DNA

Technique for Gene Transfer Materials Required:

◦A vector (used to carry the gene into the host cell)

◦A host cell (which will express the gene – make the protein)

◦Restriction enzymes◦DNA ligase

Technique for Gene Transfer A vector such as a plasmid is obtained

Bacteria carry their DNA in one large circular DNA strand

However, they posses extra DNA in the form of plasmids – circular bits of genetic material carrying ~2-30 genes

The gene we want to introduce

The DNA that we want to insert the gene into

Restriction sites

The gene is cut from its original DNA strand

The same restriction endonuclease must be used on both DNA otherwise the 2 DNAs won’t be able to bind together – there needs to be complimentary base pairing!

DNA Ligase is used to join the cut fragments of DNA together

T4 DNA Ligase – is an enzyme from T4 bacteriophage joins blunt ends together.

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Restriction enzymes act as an immune system in the bacterium. When a bacteriophage (a virus) tries to inject its DNA into the bacteria, restriction enzymes cut up the bacteriophage DNA into many fragments – thus, preventing it from doing any harm to the bacterium.

Restriction endonucleases must be able to distinguish between foreign DNA and their own DNA otherwise they would cut up their own DNA.

METHYLASES are enzymes that modify a restriction site by adding a methyl group to and preventing the restriction endonuclease from cutting it. This prevents the cell from cutting its own DNA.

Insulin is a hormone that regulates blood sugar levels by converting excess glucose into glycogen for long term storage.

Diabetics do not make sufficient amounts of insulin.

Diabetics may be required to take insulin injections.

Using restriction enzymes, the gene for synthesizing insulin is cleaved out of DNA and inserted into the DNA plasmid of a nonharmful bacteria.

DNA ligase is added to the bacteria. The bacteria is also given the necessary

amino acids. The recombinant DNA will express the

insulin gene and make insulin that can be collected and administer to diabetics.