Studying and Manipulating Genomes Chapter 15

How are Genetic changes produced? Natural processes Mutation, variation Humans have been changing the genetics of many species for thousands of years Artificial selection of plants and animals Humans changing genetics by combining genes between species Genetic Engineering

Purpose of Genetic Engineering Cure hereditary diseases Engineer livestock to Provide more nutrients Make vaccines, vitamins Engineer crops to Produce pesticides Increase productivity Better nutrients Basic research Investigate basic genetic processes Reconstruct life’s evolutionary history Devise counterattacks against rapidly mutating pathogens

First Recombinant DNA Paul Berg and associates were first to make recombinant DNA in 1972 Fused fragments of DNA from one species into the genetic material from another Allowed them to isolate and replicate subsets of DNA from any organism

Tools for Constructing Recombinant DNA Restriction Enzymes Hamilton Smith was studying Haemophilus influenzae discovered bacteria have an enzyme that chops up viral DNA. Will cut at a specific site on a specific sequence of DNA. The section has to be a palindrome! Ex. GAATTC is cut by EcoRI The same enzyme is used for both the DNA fragments so the overhanging ends are identical and complementary Ligase used to glue them together

Making Recombinant DNA

Tools for Constructing Recombinant DNA Plasmids as Vectors Small circular DNA pieces, can be used to transfer foreign DNA to bacteria Why: So that the gene can be expressed and studied Can also be inserted into eukaryotic cells directly Ex. Humans, plants etc.

Making Recombinant DNA Recombine DNA to be studied with a Plasmid DNA. Transform bacteria with this plasmid. Can be studied and manipulated.

Tools for Constructing Recombinant DNA PCR – Polymerase Chain reaction Many rounds of DNA replication in a test tube to generate thousands of copies of a DNA segment. Used for forensics Insertion and cloning sequencing of DNA

Polymerase Chain Reaction

Polymerase Chain Reaction

Polymerase Chain Reaction

Polymerase Chain Reaction DNA replication in a tube! Sequence to be copied is heated to break H bonds Primers are added and bind to ends of single strands Taq DNA polymerase uses free nucleotides to create complementary strands Repeat Steps 1-3 Doubles number of copies of DNA at each round

Tools for Constructing Recombinant DNA Gel Electrophoresis Technique by which molecules of different sizes are separated and analyzed Separated by electric charge - DNA is negatively charged and will migrate towards positive electric field Rate of migration depends on size of molecule +

Tools for Constructing Recombinant DNA T C C A T G G A C C T C C A T G G A C Tools for Constructing Recombinant DNA T C C A T G G A T C C A T G G T C C A T G Sequencing DNA T C C A T T C C A Fluorescent labeled and fragmented DNA placed on gel Fragments move off gel in size order; pass through laser beam Color each fragment fluoresces is recorded on printout electrophoresis gel T C C T C one of the many fragments of DNA migrating through the gel T one of the DNA fragments passing through a laser beam after moving through the gel T C C A T G G A C C A

Uses of genetic engineering: The Human Genome Project Goal – Use these tools to map the entire human genome Initially thought by many to be a waste of resources Process accelerated when Craig Venter used bits of cDNAs as hooks to find genes Sequencing was completed ahead of schedule in early 2001

Human Genome

Uses of genetic engineeringDNA Fingerprinting Unique array of DNA fragments Inherited from parents Even full siblings can be distinguished from one another by this technique

Analyzing DNA Fingerprints DNA is stained or made visible by use of a radioactive probe Pattern of bands is used to: Identify or rule out criminal suspects Determine paternity