Genetic Technologies Chapter 8

Genetic Engineering Intent of altering human genome Introducing new genetic material into

genome Insulin

Recombinant DNA DNA that contains genes of two species How?

› Restriction enzymes – cut out desired gene› Occur naturally in prokaryotic cells › Recognize specific recognition sites – 4 to 8 base pairs› Recognition sites are palindromes › Cuts gene (digests) in one direction only› Creates restriction fragments

Process – Restriction Enzyme

Locates recognition site (Top Strand)

Cuts the DNA backbone

Locates recognition site (Bottom Strand)

Cuts the DNA backbone

DNA separates

Sticky Ends or Blunt Ends

Sticky ends – zigzag cuts in strand Blunt ends – straight cut across strand

Putting Fragments together DNA ligase – sticky ends T4 DNIA ligase – blunt ends

› forms phosphodiester bonds in DNA

Plasmids

Small circular pieces of DNA found in bacteria Used as vectors for recombinant DNA (artificial) Restriction enzymes used to isolate specific

gene are used to cut plasmids

Process Plasmids and DNA

fragments are placed in same solution

Anneal DNA ligase is used

to form phosphdiester bond

Recombinant DNA introduced into host cell

DNA is cloned

Restriction Maps Diagrams that show all recognition sites on

a specific plasmid and distances in base pairs

Shows which restriction enzyme should be used

Allow scientists to determine which plasmids will work the best for cloning experiments

Transformation Cells that receive foreign DNA Bacterial cells sometimes will not take up a plasmid Bacteria are placed in ice water bath containing

CaCl₂ Solution is heated and cooled repeatedly disrupting

plasma membrane of bacteria allowing plasmid to enter

Solution is kept at 37⁰C to stabilize and grow

Identifying Bacteria Clones with Target Genes

Hybridization – identify cells that contain recombinant DNA

Identified using a hybridization probe – short single stranded complementary DNA molecule

Once identified bacteria can be grown in huge quantities (commercial use)

PCR Polymerase Chain Reaction

› Increase number of DNA copies from a single biological sample in a few hours

› Only specific regions of a chromosome are replicated

Process› Denaturation› Annealing› Elongation

Taq polymerase is used to put strand together

Isolated from bacteria that live in hot springs

Gel Electrophoresis

Technique used to separate fragments of DNA (PCR)

Genetic Engineering Biopharming

› Pharmaceutical products produced on large scale › Organisms are genetically engineered to produce

a specific protein › Ability to make new protein is passed on to

offspring

Genetic Engineering

Transgenic Organism (genetically modified organism, GMO)› Organisms that contain one or more genes from

another organism

Why use Genetic Engineering? Cost Larger organisms can

produce larger molecules

Better versions of organisms

80% of Canadas Canola crop is GM

Knockout Mice

Concerns?

Gene Therapy Techniques used to replace, remove or

alter a defective gene before symptoms are expressed

Gene Therapy Germ-line gene

therapy› Genes introduced

in sperm or egg cells

› Passed on to future generations

Somatic gene therapy› Genes introduced

into body cells (not sperm or egg)

› Will not be passed on

Genetic Screening To detect any mutation in DNA that is

associated with a genetic disorder (huntingtons disease)

Aminocentesis› Prenatal screening

Cord Blood (CBE) Source of hematopoietic (blood forming)

stem cells Can be used to treat and cure disease and

other conditions