Genetics 3000 Week 10 Notes
Genetics 3000 Week 10 Notes 85033 - GEN 3000 - 002
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85033 - GEN 3000 - 002
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This 5 page Class Notes was uploaded by Lisa Blackburn on Sunday April 17, 2016. The Class Notes belongs to 85033 - GEN 3000 - 002 at Clemson University taught by Kate Leanne Willingha Tsai in Fall 2015. Since its upload, it has received 36 views. For similar materials see Fundamental Genetics in Biomedical Sciences at Clemson University.
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Date Created: 04/17/16
Chapter 17: 1. Recombinant DNA Technology: set of molecular techniques used for locating, isolating, altering, and studying DNA segments. a. Allows for creation and manipulation of different sequences of DNA from different sources (organisms) b. Instrumental in developing: i. More sensitive crime scene forensics ii. Transgenic plants/animals iii. Pharmaceuticals iv. Wholegenome sequencing c. Biotechnology: technological application that uses biological systems 2. Restriction Enzyme: recognizes specific nucleotide sequence, restriction site, and makes a double stranded cut a. restriction site: usually about 58 base pair long b. We can use these enzymes to cut the DNA that we want to cut since it will recognizes bases and then cuts within the bases. They are powerful because it doesn’t care what DNA it is cutting. c. They are palindromic, meaning that they are the same forward and back, the strands are complementary to each other d. When the restriction enzyme makes a cut, it is in a defined spot and will cut there every time i. Cohesive ends: this is a sticky/adhesive overhang cut ii. Blunt end: cuts straight down and is not sticky/adhesive e. Will not distinguish between type of DNA, if sees its recognition site, it will cut there i. Can cut DNA from two different organisms, and due to sticky ends, these DNA segments can stick together 3. Gene Cloning: amplification of a specific piece of DNA a. Mostly in bacterial cells b. Cloning vector: stable, replicating DNA molecule which can attached to foreign DNA and be introduced into a cell c. Needs to be put into a cell and be stable within it, or the cell will degrade it. d. Needs to have: i. Origin of Replication: needs to be able to replicate itself inside the cell ii. Selectable Markers: to be able to see if inside the cell or not iii. Unique Reactive enzyme sites: so it will not get diced up by the cells restrictive enzymes 4. Plasmids: replicate independently of bacterial chromosome a. Perfect to be a cloning vector: i. Stable ii. Own origin of replication iii. Can be manipulated to do what we want them to do iv. Needs to be put into cell through transformation 1. Transformation: DNA is the environment is taken up by the cell b. We want sticky ends, not blunt ends, when restrictive enzymes cut c. Selectable marker around the cloning site is needed to help us tell if the cell is transformed or not i. Blue light screening: uses the lacZ gene, if the gene is intact, then it will cause a blue pigment to be, if the insertion is inside it then the cell will not be blue 5. DNA Libraries: collection of clones containing all of the DNA fragments from one source (this is what we create) a. Genomic library: consists of many overlapping fragments of the genome, with at least one copy of every DNA sequence in an organism’s genome b. cDNA library: contains DNA sequences only from genes that are transcribed c. Several clones may contain all or parts of your gene in interest, so how do you find the right clone? i. Screen the library to isolate only the clones that contain your gene of interest d. Probe: DNA or RNA complementary to sequence from a gene of interest, it is labeled 6. Polymerase Chain Reaction (PCR) a. Need: template, primer, nucleotides, polymerase b. Stages: denaturation, annealing, extension c. Cell is replicating a piece of DNA through the bacteria (original method) i. If it is done without the bacteria, then we can speed it up d. With PCR we need a template, something to start with, a piece of DNA we want to amplify e. Also needs nucleotides and new bases, polymerase to read and copy it, and primers f. Denaturation: heat up the DNA causing the bonds to break g. Annealing: set it up in the right place, cool it down for the Hydrogen bonds to form h. Extension: polymerase will attach to primers and use 3’OH and starts to copy i. Requirements: i. Template: any nucleotide source ii. Primers: identifies target and provides a starting point iii. Nucleotides: for new strand synthesis iv. Polymerase: to synthesize the DNA j. Stages: i. Denaturation: separate doublestranded DNA template. Heat to 9295 degrees C ii. Annealing: primer binding. Cool to 4575 degrees C iii. Extension: DNA polymerase synthesizes new DNA strands. Heat to 65 75 degrees C k. Happens very quickly, and exponential l. Limitations: i. Primer synthesis: need to know something about the sequence ii. Contaminating DNA iii. Accuracy iv. Amplification size v. Machines are used to heat and cool DNA as needed vi. Stable polymerase and machines allows for PCR to happen a lot and easily vii. PCR is a strong technique and is simplified, but you have to make primers if you want a specific piece amplified to anneal that piece of DNA 7. Reverse Transcription PCR: beings with RNA template. Start with step that reverse transcribes, then stays as regular PCR a. Real time PCR: how much are we getting out of each cycle? Measures gene expression levels i. Cycle of PCR and then take a picture then another cycle and then another picture. Figure out how much product is being formed from each cycles, allows to measure gene expression b. Start with lots of copies of DNA one round of amplification there is faster timing with high expression. Low expression has few strands of RNA and takes longer before having lots of amplification. Gene expression levels are measured, and the number of times the sequence is present in the genome c. Did it work? i. Gel electrophoresis: separating the DNA based on size 8. Probe: DNA or RNA complementary to sequence from a gene of interest; labeled a. Edwin Southern: Southern Blot process of transferring DNA fragments from gel to membrane i. Northern Blot: RNA ii. Western Blot: protein b. Takes a particular sample and look at each one individually i. Combine gel electrophoresis and digest the DNA with restriction enzymes. ii. Gets DNA fragments (from restrictive enzyme cuts), let it run out to see based on size iii. Do blotting plot and transfer to membrane, get DNA stuck to membrane (but has to be separated out by size first), get transferred over to membrane and relies on probes (DNA or RNA but has to be complementary and labeled to know where it should stick) 9. In Situ Hybridization: probes are used to determine chromosomal location a. Visualized in the cell (in situ) b. Fluorescence ISH (FISH) c. Can be used to probe tissues for a particular mRNA d. Which cells are they being expressed in and what stage of development? 10. Sanger Sequencing: uses dideoxyribonucleoside triphosphate which causes elongation to stop a. Most popular b. Have to have a 3’OH group in order to connect and be useful c. Dideoxy (missing both oxygens), if no 3’OH group it says it is done d. Keeps from extending to another piece of DNA 11. KNOW HOW TO READ GELS a. Template DNA: want to know what bases are there, give primer, give polymerase, split into 4 different tubes (has everything in the tubes), add in some of didoxy bases (dideoxy C, G, and T) but in the tubes there are copies of all regular DNA bases already b. Reads template and puts in new bases c. The fragments, when it is time to choose, can choose the didoxy base or the regular base, given different size fragments that are separated by size d. Polymerase can only make DNA 5’ to 3’ e. Sequence made for copy piece of DNA smallest is 5’ end, biggest is 3’ end f. The template is antiparallel and complementary Chapter 18: 1. Genomics: field of genetics that attempts to understand the content, organization, function, and evolution of genetic information contained in whole genomes a. Structural genomics: studies the organization and sequence of genetic info contained within a genome b. Genetic maps: based on recombination frequencies c. Physical maps: based on direct DNA sequence i. Map based sequencing ii. Whole genome shotgun sequencing d. Bioinformatics: emerging field consisting of molecular biology and computer science that centers on developing databases, computersearch algorithms, gene prediction software, and other analytical tools i. at the point where data is created but is used without the use of computer programs ii. all from info from bioinformatics: sequencing the entire genome 2. Comparative Genetics: compares similarities and differences in gene content, function, and organization among genomes of different organisms a. Prokaryotes: amount of DNA ranges from 490 kb to 9,105 kb i. E. coli: approx.. 4.8 million base pairs ii. This makes the comparison between how big the genome is and how it relates to the base pairs iii. One gene is about 1,000 base pairs b. Eukaryotes: genome is larger than the prokaryotes i. No relation between genome size and complexity ii. No correlation between genome size and number of genes iii. Has move/larger genomes than most prokaryotes but there is no relationship to the genes c. Gene deserts: large area with no known genes d. Transposable elements: ~45% of DNA in human genome e. Protein diversity: modifications greatly increase number of proteins f. Number and length of introns increases in more complex eukaryotes g. Reference genome: several different pieces of genomes combined i. all humans are 99.9% the same ii. our genomes are the same, the difference is in the molecular markers of each individual 3. Functional Genomics: determining what the sequences do a. Transcriptome: all the RNA molecules transcribed from a genome i. All the transcripts, what is being expressed, just like the library, its unique to each tissue and each time point, very dynamic because it is being expressed b. Proteome: all the proteins encoded by the genome i. What has been translated into a protein, very dynamic, changes from one tissue to the next c. The biggest piece of the genome is unknown 4. Microarrays: monitors gene expression in different tissues or under different conditions a. Nucleic acid hybridization b. Microarray is looking into the transcriptome. The probes (dots) carry different amounts of expression. You can detect gene expression whether it is there or not for MANY genes with just one sample 5. Proteomics: study of the proteome (complete set of proteins found in a given cell) a. what proteins are actually being made, 2 major (mass spectrometry and protein microarrays) b. Mass spectrometry: method for precisely determining the molecular mass of a molecular i. Masstocharge (m/z) ratio c. Protein microarrays can be used to analyze proteinprotein interactions d. Twodimensional gel electrophoresis (2DGE): separates proteins based on isoelectric point and then size i. Separate proteins first, charged proteins separate based on charge and size ii. Each dot is a different protein iii. Look to see if they were the same or if it was made effective or ineffective protein e. Systems biology: where bioinformatics comes in, tells computer what to look for and how to find relationships i. Genomic information: starting to say try to look at how genes and genomes are all related
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