BIOTECHNOLOGYApplications and Basic Technology

Recombinant DNA technology

Recombinant DNA technology: set of techniques for recombining genes from different sources and transferring into cells where it may be expressed

Restriction Enzymes

Occur in bacteria where they protect against intruding DNA

Involves restriction; foreign DNA is cut into small segments

Cut at specific points on small segments called specific recognition sequences

Several hundred restriction enzymes & ~ 100 different specific recognition sequences

Cuts DNA in staggered manner, resulting in single-stranded ends, called sticky ends

Sticky Ends v. Blunt Ends

Creates restriction fragments

Used in lab to join DNA pieces from different sources

Temporary bonds held by weak H bonds

Can be made permanent by adding DNA ligase

Gel Electrophoresis

used to separate either nucleic acids or proteins based upon molecular size, charge and other physical properties

used to identify segments by looking at banding pattern when cut with restriction enzymes

DNA fragments can be isolated, purefied, and then recovered from gel

Electrical current is used to move charged particles at different rates

RFLP Analysis (restriction fragment length polymorphisms)

gel electrophoresis of restriction fragments results in characteristic banding pattern

Each band corresponds to DNA restriction fragments of certain length

Different alleles of gene result in dissimilar banding patterns

Similar patterns result when noncoding segments of DNA are used as starting materials

Fragments move different distances based on base pair length, size (how compact)

Used to match DNA from different sources

Recombinant DNA

Use restriction enzymes to alter DNA Incorporate DNA into new organism to

create a GMO (genetically modified organism)

Ex: Super Salmon, Flavr Savr Tomato Super Salmon ABC Special

Polymerase Chain Reaction

Technique used to amplify (copy many times) DNA in vitro (inside the cell)

1. DNA is incubated with special primers & DNA polymerase

2. billions of copies of the DNA are produced in a few hours

3. PCR is highly specific; primers determine sequence to be amplified

4. only minute amounts of DNA are needed