Genetic Technology

Learning Objectives By the end of this class you should understand: The concept and mechanics of cloning The purpose and methodology of DNA restriction Methods relating to keeping libraries of genes The purpose and mechanics of PCR The purpose and mechanics of a Southern blot and microarray analysis

Genetic Technology So far material has focused on the existing system of DNA Have to know the system before you can propose manipulating it!

Cloning Cloning has been the subject of fiction long before it became fact Cloned cells are genetically identical Cloned individuals do NOT share thoughts or memories since those are not genetic

Cloning Methods Embryo Splitting Nuclear Fusion Essentially intentional creation of identical twins/triplets Nuclear Fusion Insertion of a nucleus into an enucleated egg and then implanting the egg into a surrogate mother

Enucleated Egg Removal of a nucleus from an egg cell creates an enucleated egg This is useful because it still contains all the cytoplasmic factors needed to develop into an embryo Fusion was originally performed with an electrical shock, now performed with a microneedle

Cloning Process

Why Clone? An animal that has a particular advantage can be bred with other animals but regression to the mean will likely occur Cloning produces entire herds of cattle or sheep that are the most effective at producing milk/wool/etc Downside: clones are all vulnerable to the same diseases!

Gene Cloning Another major technique is to clone a section of DNA rather than an entire organism Known as recombinant DNA technology Re-combining (mixing- and-matching) DNA Required to place individual genes into living things

Key Recombinant DNA Tool The most useful tool in cloning genes is called a restriction enzyme Restriction enzymes cut DNA in a distinctive pattern that leaves “sticky ends” Only cuts very precise sequences of DNA

Restriction Enzymes Originally discovered in bacteria as virus protection Useful now to insert pieces of DNA into other pieces of DNA

Restriction Enzyme Activity If two DNA strands with the same restriction sequences are mixed and restricted they will then pair indiscriminately To reseal the DNA, the enzyme DNA ligase must be used Same enzyme that attaches Okazaki fragments!

Bacterial Plasmids Bacteria have a bacterial chromosome with all their DNA, but they may also carry small “bonus gene” carriers called bacterial plasmids May carry genes for toxins or antibiotic resistance May be traded to other bacteria May be injected into bacteria

Bacterial Plasmid Use Different plasmids have restriction enzyme sites, and may have genes inserted into them using restriction enzymes In this manner, bacteria may be induced to express any gene!

pBR322 Standard plasmid for use in gene cloning Has gene inserted via restriction enzymes Injected into bacteria Becomes a vector for cloned gene Sometimes include an antibiotic resistance gene in vector to weed out other bacteria

Recombinant Plasmids

Storage of Cloned Genes Genes can be stored in a genomic library of bacterial colonies Genes can be located using a probe made of DNA which matches only a certain gene If bacterial colonies are mixed together, a radioactive probe can identify the desired colony

DNA Probe

Storage of Chromosomes Larger sequences of DNA may be stored also Plasmid can only hold one gene Yeast Artificial Chromosome is useful for storing large sections of DNA Up to 1 million bases (several hundred genes)

Polymerase Chain Reaction Polymerase chain reaction (PCR) is a gene cloning technique invented in the 1980s Produces billions of copies of a gene in hours Essentially, DNA strands are separated and mixed with free DNA bases and Taq DNA Polymerase DNA Polymerase from a hot springs bacterium so it can handle the heat

PCR Diagram

PCR Part 2

PCR Part 3

PCR Process Demonstration: http://www.youtub e.com/watch?v= _YgXcJ4n-kQ

PCR Applications If a single hair is taken from a crime scene, is there enough DNA to analyze? If not, use PCR! DNA can be cut with restriction enzymes into different lengths and run in a Southern Blot Each bar is a different length of DNA Smaller = faster

Southern Blot Identity If two identical strands of DNA are restricted by the same enzyme they will produce the same pattern Smaller pieces move faster than larger ones This is how DNA fingerprinting works!

Southern Blot Results DNA is bound with radioactive probes or luminescent DNA stain Result shows different patterns because different DNA has different restriction sites

Microarray Analysis Microarrays are used to study gene expression rather than gene presence Southern blot will show same results for all cells from a particular human Even cancerous cells will look similar

Microarray Procedure Each well contains complementary DNA (cDNA) that matches a mRNA for a gene When a tissue sample is lysed, its mRNAs will bind to the cDNA of each well Different cDNAs are labeled different colors

Microarray Analysis When two different tissues are put in simultaneously, each is associated with a particular dye For example, normal cell expression is green and cancer cell expression is red This shows which genes are no longer expressed and which are newly expressed in cancer cells

See you tomorrow!