Biotechnology & Recombinant DNA

What is biotechnology?  Using living microorganisms or cell components to make products Often via genetic engineering and using recombinant DNA

Recombinant DNA technology:  Insertion or modification of genes to produce desired proteins.

What is recombinant DNA?  Inserting foreign DNA into a bacterial cell  Use restriction enzymes, plasmids, ligase and bacterial host  Restriction enzymes Defense against viruses  Clone gene or make gene product

How is recombinant DNA made?  Recall: happens naturally Transposons  Researchers can also produce it Use bacterial plasmids or viruses (bacteriophages)  Uses Bacteria make insulin yeast help make components of Hepatitis B vaccine

What are restriction enzymes?  Naturally occur in bacteria Bacteria use these to combat viral infection Bacteria DNA uneffected because some is methylated  Sequence- specific  Restriction enzymes Restriction enzymes  Cloning animation Cloning animation This can be used as a vector…

What is a vector?  Different from a disease vector!  plasmid animation plasmid animation  Plasmid or virus Used to insert DNA into host cell Must be able to self-replicate! Must be small so not fragile  Both types of vectors can allow researchers to clone DNA But there’s another approach to DNA amplification…

PCR: making copies of DNA

What is PCR? Figure 9.4 (1 of 2) Stands for _____________ Need primers of about 20 bp to start DNA polymerase doesn’t start reaction, only lengthens Primers recognize regions which flank target gene Ingredients primers DNA polymerase from TAC (Thermus aquaticus) Individual nucleotides Can you go over that one more time? …PCR animationPCR animation

What is PCR? Figure 9.4 (2 of 2) Stands for _____________ Need primers of about 20 bp to start DNA polymerase doesn’t start reaction, only lengthens Primers recognize regions which flank target gene Ingredients primers DNA polymerase from TAC (Thermus aquaticus) Individual nucleotides Can you go over that one more time? …PCR animationPCR animation

Why would you use PCR?  Detect small amounts of DNA Can you think of examples?  Get in groups and discuss!  Forensics  Infectious agents  Gene mapping Human Genome Project  Taxonomy and systematics studies  Cancer and study of other human diseases  Sequencing of rRNA and mRNA via cDNA

DNA fingerprinting

Let’s take a brief look at some of these…

What is gel electrophoresis?  Sorting DNA segments by size  DNA fingerprinting Restriction enzymes create restriction fragment length polymorphisms (RFLPs) Gel electrophoresis animation

What is Southern blotting?  After gel electrophoresis  Filter paper blots DNA off  Radioactive probes added  Autoradiography pinpoints sequence  Southern Blot animation Southern Blot animation

The scientific applications  Understanding of DNA  Sequencing organisms' genomes  DNA fingerprinting for identification Figure 9.17

Why E. Coli is used!  Used because it is easily grown and its genomics are known  Need to eliminate endotoxin from products  Cells must be lysed to get product

Inserting Foreign DNA into Cells  DNA can be inserted into a cell by Transformation Electroporation Protoplast fusion Microinjection Gene gun

What is RNA Interference (RNAi)? Figure 9.14 RNAi animation

Cloning

How do researchers get the DNA they want to clone?  Gene libraries are made of pieces of an entire genome stored in plasmids or phages.  cDNA is made from mRNA by reverse transcriptase.  Synthetic DNA is made by a DNA synthesis machine.

So what’s the problem?  Fine for prokaryotic DNA  Problem with eukaryotic DNA…  Eukaryotic DNA has introns… Must make complementary DNA (cDNA) Use reverse trasncriptase  cDNA animation cDNA animation  Now DNA can be inserted…

What do they do with the cloned DNA?  Lots of different things!

Is this the only way to “look” at DNA?  No—we can sequence it, too!  Random shotgun sequencing  Start with a whole genome or a large piece of the DNA (a BAC). BAC-bacterial artificial choromosme  Shear the DNA into many different, random segments.  Sequence each of the random segments Put the pieces back together in original order

What are bioreactors?  Using bacteria to produce gene products Insulin: diabetes Human growth hormone Cellulase (break down cell wall for animal feed) Factor VIII: hemophilia

What are plant GMOs?  Genetically modified organisms Transgenic plant or animal  Bioreactors filled with these  Plants Cotton, corn, potato to make pest resistant Soybeans resistant to common herbicide Some corn, cotton are herbicide and pest resistant Could produce human hormones, clotting factors, antibodies on seeds in future

What are animal GMOs?  Foreign genes into embryos  produce animals that manufacture human hormones, etc. = gene pharming Blood clotting factor goats Sheep milk with human alpha-1- antitrypsin (used to treat heritable emphysema)  1997, Dolly  Since then, cloned sheep, cows, goats, mice  Humans: moratorium

What is gene therapy?  Insertion of genetic material into human cells to treat a disorder Use retrovirus to insert normal gene into cell  Healthy genes to make up for faulty genes Severe combined immunodeficiency syndrome 1990, girl received normal gene in white blood cells  Using genes to treat other illnesses