Chapter 20: Part 1 DNA Cloning and Plasmids DNA Technology and Genomics

Biotechnology DNA technology has launched a revolution in the area of biotechnology The manipulation of organisms or their genetic components to make useful products Example: gene cloning Usually uses bacterial plasmids

Figure 20.2 Bacterium Bacterial chromosome Plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest DNA of chromosome Recombinant bacterium Protein harvested Basic research on protein Copies of gene Basic research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth Protein expressed by gene of interest 3 Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes

How do You Make Recombinant DNA? Use bacterial restriction enzymes Called restriction endonucleases Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites

Restriction enzymes Where are restriction enzymes found? In bacterial cells, to help fight viruses/ foreign DNA What is a restriction site? The area on DNA that the restriction enzyme recognizes Every time this DNA sequence occurs in bacterium’s own DNA, methyl groups (-CH3) are added  prevents restriction enzyme from working. When foreign DNA (ex: a bacterial virus) enters bacterium, bacterium’s restriction enzyme cut phage’s DNA into pieces. NOT all bacteria have restriction enzymes, many do! But there is a wide variety of restriction enzymes that have been discovered and continue to be discovered

Restriction Sites Many different kinds EcoRI, BamHI, HindIII, HaeIII, etc. Restriction site is like a palindrome A palindrome is a word or phrase which reads the same in both directions. These enzymes make “blunt” and “sticky” ends. What is the difference?

Restriction Enzymes Enzymes usually make many cuts in a DNA molecule Yields a set of restriction fragments Most useful restriction enzymes cut DNA in a staggered way Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of any other DNA fragment DNA ligase is an enzyme that seals the bonds between restriction fragments

Different restriction enzymes that can be used

Restriction Enzymes http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html ANIMATION #1

Restriction Enzymes: Proteins that cut the DNA in a specific place Recombinant Plasmid

Transformation  bacteria takes in plasmid from environment

Cloning The original plasmid is called the cloning vector DNA molecule that can carry foreign DNA into a cell and replicates there After insertion of the gene of interest into the plasmid the plasmid is called recombinant DNA Recombinant Plasmid

How do you clone a gene using a plasmid?

Steps in DNA recombination Isolate “needed: DNA gene & plasmid Cut (“digest”) BOTH DNA samples (gene of interest and plasmid) with the SAME restriction enzyme Mix DNA’s together- they join at sticky ends via DNA base pairing Add ligase to seals up sticky ends **Product is the recombinant DNA Add CaCl2 & bacteria (makes cell competent) Collect products (proteins) of cloned gene  (Ex: insulin, Human Growth Hormone)

Steps in Cloning a Gene Using a Plasmid http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html ANIMATION #8

Why make DNA recombinants? There are 3 main reasons for creating recombinant DNA to create a protein product to create multiple copies of genes to insert foreign genes into other organisms to give those organisms a new trait Make DNA fingerprints Recombinant DNA is used widely today to create large amounts of protein for treating illnesses Ex: In 1982, insulin became the first recombinant DNA drug to hit the market

Plasmid Cloning http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html

Identifying the gene of interest How would you identify different genes? Use different probes

2 ways that we know the cell clones carry the recombinant plasmids? Look for the specific product (protein) that the foreign DNA codes for Is the bacteria making this protein? If yes, the cloning worked! Use a “nucleic acid probe” A single stranded nucleic acid molecule used to “tag” a specific nucleotide sequence in a sample Hydrogen bonding is used!