BIOTECHNOLOGY• DNA is now being easily manipulated.• Molecular biologists analyze and alter genes

and their respective proteins.• Recombinant DNA is DNA from one source

organism being put into the DNA of a host organism.

• The tools the scientists use are very specific to the DNA or the environment they work in.1. the DNA first has to be cut out of the source organism2. the DNA has to be isolated3. the DNA can then be introduced into the host DNA

1) Cutting Out DNA: RESTRICTION ENDONUCLEASES / ENZYMES

Naturally occurring enzymes that act like a pair of molecular scissors to cut DNA at a specific nucleotide sequence called a recognition site .

Hamilton Smith, John Hopkins University, won the Nobel Prize in 1978 for discovering restriction enzymes in bacteria (HindIII)

He found their main purpose was to cut foreign DNA that tried to invade a bacterial cell (ie DNA from a virus).

Restriction enzymes are named according to the bacteria from which they originate.

BamHI is from Bacillus amyloliquefaciens, strain H. The I indicates it was the first endonuclease isolated from that strain.

In the lab, restriction endonucleases have become a common tool allowing scientists to cut DNA in a predictable and precise manner.

The restriction enzyme EcoRI binds to 5'-GAATTC-3' / 3'-CTTAAG-5Palindromic: both strands have the same sequence when read in the 5' to 3' direction.

recognition sites are usually 4 – 8 base pairs in length. Table 1 p 279 – examples

EcoRI finds this recognition site and breaks the phosphodiester bond between G and A, then it pulls apart the two strands by breaking the H-bonds between the complementary base pairs.

Produces what are called sticky ends (unpaired nucleotides at each end).

• Other restriction enzymes like AluI produce blunt ends, ends with no overhang.

• Sticky ends are usually more helpful to molecular biologists as they can easily be joined with other DNA fragments cut by the same restriction enzyme. Blunt ends are harder to fuse to a foreign DNA molecule.

• P 281 #1-5

A host must protect its own DNA from endonucleases.

• Methylases are enzymes that place a methyl group (CH3) on a recognition site for a restriction enzyme which prevents the restriction enzyme from cleaving the DNA at that spot.

• Host DNA is methylated, but foreign DNA is not, so it is usually cut by the host cell's restriction enzymes.

2) ISOLATING DNA FRAGMENTS: GEL ELECTROPHORESIS

• Restriction endonucleases cleave the DNA into smaller fragments

• Gel electrophoresis is used to isolate the required gene segment from the rest of the DNA

• The fragments of DNA will be run through a porous agarose gel using electricity.• The fragments of DNA are pulled

through pores in the gel due to their negative charge.

• Smaller fragments will move faster than larger because they can fit through the pores better.

http://content.answers.com/main/content/wp/en/a/ab/Agarose_Gel_Electrophoresis.png

• STEPS:• Solutions of these fragments are placed in wells (depressions at one end of

the gel)• The DNA is mixed with a loading dye so it will be seen as it moves through

the gel.

• Markers are usually put in the first well. These are pieces of DNA whose size is known. They help determine the length of the unknown DNA fragments.

• The gel is submerged in a buffer solution and then connected to a power source.

• The negative end of the power source will be at the top and the positive end at the bottom. DNA is negatively charged, it will move away from the negative electrode to the positive electrode.

• The power source is only left on for a set amount of time so that the fragments don't all move to the end – you want them separated .

http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.gif

VIEWING THE GEL

• After the power source is turned off, the gel is stained with ethidium bromide, which will cause the gel to fluoresce under UV light.

• Under the UV light the bands of the DNA fragments can be seen and the researcher is able to compare samples from various sources or isolate a DNA fragment that they want to purify and use in their experiments.

http://www.mcps.k12.md.us/departments/intern/stp/images/gel_electrophorsis.jpg

http://www.life.uiuc.edu/molbio/geldigest/fullsize/geldraw.jpg

3) INTRODUCING FOREIGN DNA INTO A HOST: PLASMIDS and TRANSFORMATION

• Plasmids are small (1000 to 200 000 bp in length), circular DNA molecules independent of the bacterial chromosome.

• Plasmid DNA can be replicated using the cell's machinery independently of the chromosomal DNA.

• Beneficial because they often contain important genes– Antibiotic resistance,

heavy metal protection

TRANSFORMATION• The recombinant DNA is then

introduced into a bacterial cell. Sometimes a host cell must be manipulated to take up the foreign DNA plasmid.

• Transformation: introduction of foreign DNA (usually by plasmid or virus) into a bacterial cell.

• Host cell: cell that has taken up foreign plasmid or virus and whose cellular machinery is being used to express the foreign DNA.

• Competent cell: cell that readily takes up foreign DNA.

• When the bacteria replicates the recombinant DNA plasmid, the new gene product will be formed multiple times (ie. the gene is cloned).

• Cells with successful transformation will be grown in culture to produce multiple copies (clones) of the incorporated recombinant DNA.

PCR – another means of copying DNA in large numbers

• stands for Polymerase Chain Reaction, developed in the late 1980's

• amplification of a DNA sequence by repeated cycles of strand separation and replication in the laboratory (DNA photocopying).

• Copies exponentially increase; after about 30 cycles of PCR, more than one billion copies of the sequence will exist.

• Does not require a plasmid.• Useful for forensic criminal investigations,

medical diagnosis, genetic research.• Diagram p. 297