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Chapter 3 Recombinant DNA Technology and Genomics Agarose Gel Electrophoresis Electrophoresis is a molecular technique that separates nucleic acids and proteins based on: Size and +-+Charge+-+ Agarose Gel Electrophoresis DNA is a negatively charged molecule and therefore is attracted to positive charges. Agarose Gel Electrophoresis Agarose provides a matrix through which DNA molecules migrate. Larger molecules move through the matrix slower than small molecules The higher the concentration of agarose, the better the separation of smaller molecules Agarose Gel Electrophoresis How to make an agarose gel: 1. 2. 3. 4. Weigh out a specified amount of agarose powder. Add the correct amount of buffer. Dissolve the agarose by boiling the solution. Pour the gel in a casting tray. 5. Wait for the gel to polymerize. Agarose Gel Electrophoresis How to make an agarose gel: 6. Place gel in chamber and cover with buffer 7. Add loading dye to the sample 8. Load sample on to the gel. Agarose Gel Electrophoresis How to make an agarose gel: 9. Stain the gel 10. Take a picture of the gel 11. Analyze results Agarose Gel Electrophoresis Electrophoresis Animation Recombinant DNA Recombinant DNA technology Allows DNA to be combined from different sources Also called genetic engineering or transgenics Recombinant DNA Vector – DNA source which can replicate and is used to carry foreign genes or DNA fragments. Recombinant DNA – A vector that has taken up a foreign piece of DNA. Restriction Enzymes Restriction enzyme – an enzyme which binds to DNA at a specific base sequence and then cuts the DNA Restriction enzymes are named after the bacteria from which they were isolated. • Bacteria use restriction enzymes to chop up foreign viral DNA Restriction Enzymes Recognition site – specific base sequence on DNA where a restriction enzyme binds. All recognition sites are palindromes, which means they read the same way forward and backward. example: RACECAR or GAATTC CTTAAG • Each restriction enzyme has its own unique recognition site. • Restriction Enzymes Restriction Enzymes After cutting DNA with restriction enzymes, the fragments can be separated on an agarose gel. • The smaller fragments will migrate further than the longer fragments in an electric field. • The bands are compared to standard DNA of known sizes. This is often called a DNA marker, or a DNA ladder. Restriction Enzymes Running a Restriction digest on an agarose gel Restriction Enzymes After analyzing your results, you draw a restriction map of the cut sites. • A restriction map is a diagram of DNA showing the cut sites of a series of restriction enzymes. Restriction Enzymes Restriction Enzymes Restriction Enzymes Most restriction enzymes cut within the recognition site. When restriction enzymes cut in a zig zag pattern, sticky ends are generated. Restriction Enzymes Overhanging sticky ends will complementarily base pair, creating a recombinant DNA molecule. DNA ligase will seal the nick in the phosphodiester backbone. Restriction Enzymes Restriction Enzyme Animation Transformation Transformation – the process by which organisms take up and express foreign DNA Griffith’s experiment Transformation Bacterial Transformation Bacteria, such as E.coli, can take up and express foreign DNA, usually in the form of a plasmid. Transformation Gene cloning – using bacterial transformation to make lots of copies of a desired gene. Gene Cloning Animation Transformation Steps of Bacterial Transformation 1. Choose a bacterial host a. E. coli is a model organism i. ii. iii. iv. v. vi. vii. Well studied No nuclear membranes Has enzymes necessary for replication Grows rapidly (20 min. generation time) Inexpensive Normally not pathogenic Easy to work with and transform Transformation Steps of Bacterial Transformation 2. Choose a plasmid to transform a. Characteristics of a useful plasmid i. Single recognition site • ii. Origin of replication • iii. Plasmid only cuts in one place, so this ensures that the plasmid is reformed in the correct order. Allows plasmid to replicate and make copies for new cells. Marker genes • Identifies cells that have been transformed. gene for antibiotic resistance – bacteria is plated on media with an antibiotic, and only bacteria that have taken up a plasmid will grow gene that expresses color – bacteria that have taken up a recombinant plasmid are a different color than bacteria that have taken up a NONrecombinant vector. Transformation Steps of Bacterial Transformation 3. Prepare bacterial cells for transformation a. b. c. Treat with calcium chloride – softens the phospholipid bilayer of the cell membrane, which allows the plasmid to pass through Electroporation – brief electric pulse Directly inject plasmid into bacterial host Transformation Steps of Bacterial Transformation 4. Plate transformation on appropriate media a. Contains nutrients for bacteria and antibiotic to distinguish transformed bacteria from NONtransformed bacteria 5. Incubate plates overnight a. E.coli grows at body temp. (37 °C) 6. Analyze plates Gene Cloning Animation Gene Cloning What makes a good vector? Gene Cloning What makes a good vector? Gene Cloning How do you identify and clone a gene of interest? • BUILD A LIBRARY!! • DNA library – a collection of cloned DNA fragments from a particular organism • Can be saved for a relatively long period of time and screened to pick out different genes of interest • Two types of libraries 1. Genomic library – contains DNA sequences from entire genome 2. cDNA library – contains DNA copies of mRNA molecules expressed Construction of a DNA library Animation Gene Cloning Gene Cloning Steps to screen a library 1. Plate cells and transfer to nylon membrane 2. Lyse bacterial cells 3. Denature DNA 4. Add radioactively labeled probe that is complementary to gene of interest Gene Cloning Steps to screen a library 5. Wash off non-specifically bound probe 6. Expose membrane to x-ray film 7. Align exposed film with original plate 8. Grow cells containing gene of interest in culture. Gene Cloning Rarely is an entire gene cloned in one piece, even in a cDNA library, therefore must “walk” the chromosome until a start and stop codon are found. Sequencing Sequencing – determining the order and arrangement of G’s, A’s, T’s and C’s in a segment of DNA. Sequencing Let’s review replication….. Sequencing The Sanger sequencing method uses dideoxynucleotides to generate all possible fragments of the DNA molecule to be sequenced. deoxynucleotide dideoxynucleotide Sequencing Set up four different reactions: Sequencing Load the four reactions in different wells of a polyacrylamide gel to separate the fragments Sequencing Sequencing Animation Human Genome Project Initiated in 1990 with plan to identify all human genes • Analyze genetic variation among humans • Map and sequence genomes of model organisms • Develop new lab technology • Disseminate genome information • Consider ethical, legal, and social issues that accompany genetic research Human Genome Project Francis Collins Craig Venter Human Genome Project Consider ethical, legal and social issues • Who owns your DNA? Human Genome Project Develop new lab technology • Automated Sequencing Human Genome Project Disseminate genome information • GenBank database Human Genome Project Analyze genetic variation among humans • The genome is approximately 99.9% identical between individuals of all nationalities and backgrounds. Human Genome Project Map and sequence genomes of model organisms • E.coli • Arabidopsis thaliana • Saccharomyces cerevisiae • Drosophila melangaster • Caenorhabditis elegans • mus musculus PCR Polymerase chain reaction (PCR) A lab technique used to amplify segments of DNA "PCR has transformed molecular biology through vastly extending the capacity to identify, manipulate and reproduce DNA. It makes abundant what was once scarce -- the genetic material required for experimentations." PCR Reaction requirements Template DNA – total genomic DNA isolated from an organism that contains a target region to be amplified DNA primers - Short pieces of single stranded DNA that flank the target Taq DNA polymerase - Attaches nucleotides on the growing strand of DNA Nucleotides (GATC) – Polymerase adds complementary nucleotides to the template PCR Reactions are placed in a machine called a thermal cycler. The machine cycles through three temperatures. PCR 1. Heat samples to 94°C for a minute or so to denature the double stranded template DNA. PCR 2. Drop temperature to around 50 or 60°C to allow primers to anneal. PCR 3. Maintain temperature at 72°C for a minute or two to allow the polymerase to elongate the new DNA strands. PCR The thermal cycler repeats the denaturing, annealing, and elongating temperatures approximately 30 times. PCR Animation PCR PCR amplification is logarithmic, meaning the number of copies of the target is doubled every cycle. (2n) PCR PCR animation PCR Cloning by PCR • Design primer specific for gene of interest (must know some of the sequence) • Can use a T-vector because Taq polymerase adds an A to the 3’ end of sequence Applications of Recombinant DNA Technology Chromosomal Location and Gene Copy Number Fluorescence in situ hybridization (FISH) Chromosomal Location and Gene Copy Number Southern Blot - molecular technique where DNA is transferred onto a membrane from an agarose gel and a probe is hybridized. Southern Blot The first step in preparing a Southern Blot is to cut genomic DNA and run on an agarose gel. Southern Blot The next step is to blot or transfer single stranded DNA fragments on to a nylon membrane. Southern Blot The next step is to hybridize a radioactively labeled DNA probe to specific sequences on the membrane. Southern Blot The last step is to expose the radioactively labeled membrane to a large sheet of film. You will only visualize bands where the probe hybridized to the DNA.. Southern Blot Southern Blot Animation Studying Gene Expression Northern Blot Isolate RNA from tissue of interest Separate on agarose gel Blot onto nylon membrane Hybridize probe specific for desired transcript • Expose on film • • • • Reverse Transcription PCR (RT-PCR) • Used if RNA produced is below detection level for Northern blot • Isolate RNA from tissue of interest • Convert into double stranded cDNA • Amplify by PCR • Run on agarose gel Studying Gene Expression Real Time PCR (qPCR) • Eliminates the need for running agarose gels • Is quantitative Studying Gene Expression Gene microarray Microarray animation