Genetics Week 4 Notes
Genetics Week 4 Notes Bisc 336
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This 13 page Class Notes was uploaded by Anna Ballard on Friday September 23, 2016. The Class Notes belongs to Bisc 336 at University of Mississippi taught by Ryan Garrick in Fall 2016. Since its upload, it has received 15 views. For similar materials see Genetics in Biology at University of Mississippi.
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Date Created: 09/23/16
Lecture 10 9/16 Learning Goals At the end of this lecture you should be able to: • Explain the role of restriction enzymes (Res), vectors, and host cells in cloning of DNA fragments - Good to have lots of copies because you can do much more work with it • Know what is happening in each of the major steps in the Polymerase Chain Reaction (PCR) Recombinant DNA Artificial combination of DNA from different sources • Creation of hybrid molecules that don’t occur naturally (≠ product of crossing-over in meiosis) - E.g., gene from a fruit fly put into a bacterial plasmid o Gene from two different species that would not normally mate to create mosaic-like chromosomes • Often, is just a tool used to isolate “target DNA” and make many identical copies of it Recombinant DNA • Restriction enzymes: cut ‘target’ DNA at recognition sites • Vectors/plasmids: take up DNA from the environment (lab) - Seal ends to make complete circular piece • Competent bacterial cells: transformed, then clonal growth - Receptor sites – take plasmids up and bring them into cell and DNA replication can proceed • Downstream applications (electrophoresis, sequencing) Restriction Enzymes (REs) - Original source from bacteria as a defense mechanism against bacteriophages. • Cut DNA: “scan” DNA for specific recognition sequence, and when found, cleave both strands - Cuts in zigzag fashion • Create restriction fragments… may have ‘sticky ends’ (i.e., single stranded DNA overhang) Vectors (= plasmids) • A vehicle for transporting a restriction fragment into a bacterial cell + a replicator of the inserted DNA • Most only handle fairly small fragments (~2 kb) • Often have been designed to have specific properties that make them easier to use when doing cloning Good vectors… - Polylinker region – region that would be cut by enzymes so that it opens up to insert DNA fragment of target gene in it o it is versatile and can be cut by many different enzymes o the region is a functional gene called Lac Z gene and is involved in breaking down food and changes color of cell when it is function ing properly only works when NO inserted DNA in genes bacterial cell with this plasmid will break down food and turn blue - ampicillin resistance gene – bacteria takes this up and will be able to grow on a medium o allows bacteria cells to survive on a growth medium • Replicate themselves + inserted DNA, once in host • Have several known RE cuts sites (= versatile) • DNA inserts at a location that disrupts a color gene • carry a selectable marker, only transformed hosts survive Sticky ends and DNA ligase FIG. 17-2 • Start with cleavage with EcoRI from eukaryotic gene and one from bacterial plasmid (vector) • Fragments with complementary Tails • Annealing allows recombinant DNA molecules to form by complementary base pairing • DNA ligase seals the gap A recombinant DNA molecule • if intact, blue colonies form • when broken white colonies • host cell needs this gene to survive ampicillin treated growth medium What would happen if one of this vector failed to take up the plasmids at all? - They would die before they’d even be able to be seen because they would not be resistant to ampicillin and would die from the growth medium Transforming the Host Cell • lab strain of E.coli often used as bacterial ‘host’ • ‘competent’ = take up DNA from envt. (including recombinant plasmids)… when shocked - Heat shock or electric shock • Modified S. cerevisiae (yeast) used as host cells to study expression of cloned eukaryotic genes Cloning – summary • Host-cell chromosome • the two DNAs are ligated to form a recombinant molecule • DNA to be cloned is cut with same restriction enzyme • introduction into host cell • host cell makes copies Polymerase Chain Reaction (PCR) • Uses a DNA Pol. Enzyme (Taq) that has high copying accuracy and happily operates at high temperatures • 3-step cycle makes identical copies of ‘target DNA’ • Use synthetic primers to set boundaries of ‘target’ PCR: first cycle • Denature dsDNA: heat to roughly 95°C –> becomes ssDNA - Separating two strands of DNA • Primers: roughly 20-30bp long, synthesized commercially • have polarity: 3’ and 5’ ends • annealing: primers form bonds with ssDNA (roughly 50°C) • Extension: Taq adds to 3’ end of primer (roughly 70°C) • 1 cycle doubles number of ‘target DNA’ templates PCR: second cycle • 1 cycle has 3 parts (denature, anneal, extend) • Each cyle doubles number of template from previous cycle leads to exponential increase… 25-30 cycles usually all that’s needed Advantages (v. Cloning) • easy (no host cells) and fast (hours, not days) • uses primers rather than RE recognition sites to isolate the ‘target DNA’ (more versatile) • Requires very little DNA as starting material - E.g., forensics (single blood drop, or hair) Limitations (v. cloning) • primer design: requires some baseline information about DNA sequencing of an Organism (or close relative) • prone to contamination : tiny amount of starting DNA needed, so require several negative controls Lecture 11 9/19 Learning Goals • Know what is happening in each of the major steps in Polymerase Chain Reaction (PCR) • Understand how PCR amplicons can be assayed for DNA mutations using and restriction enzymes • Recall why RT-PCR generates ‘snapshots’ of tissue-specific gene expression Cloning: recap • cloning isn’t always perfect, sometimes the bacteria fails to take up the inserted target DNA - If something goes horribly wrong – all those bacterial cells will fail to grow because they lack the gene that makes them resistant to ampicillin Which of the following steps are involved in PCR, and correct order (within a single cycle)? A) annealing –> denature –> extension B) Denature –> duplicate –> replicate C) Polymerase –> chain –> reaction D) Denature –> annealing –> extension Polymerase Chain Reaction (PCR) • Uses a DNA Pol. Enzyme (Taq) that has high copying accuracy and happily operates at high temperatures • 3-step cycle makes identical copies of ‘target DNA’ • Use synthetic primers to set boundaries of ‘target’ PCR: first cycle • Denature dsDNA: heat to roughly 95°C –> becomes ssDNA - Separating two strands of DNA and each are available to act as a template for synthesis of new strands • Primers: roughly 20-30bp long, synthesized commercially • have polarity: 3’ and 5’ ends - Can only had nucleotides to the 3’ end of original strand - DNA polymerase is the enzyme that adds to the primers • Annealing: primers form bonds with ssDNA (roughly 50°C) • Estension: Taq adds to 3’ end of primer (roughly 70°C) • 1 cycle doubles number of ‘target DNA’ templates PCR: second cycle • 1 cycle has 3 parts (denature, anneal, extend) • Each cycle doubles number of template from previous cycle leads to exponential increase… 25-30 cycles usually all that’s needed - Before first cycle: 1 strand - After first cycle: 2 strands - After second cycle: 4 strands Advantages (v. Cloning) • easy (no host cells) and fast (hours, not days) • uses primers rather than RE recognition sites to isolate the ‘target DNA’ (more versatile) • Requires very little DNA as starting material - E.g., commonly used in forensics (single blood drop, or hair) Limitations (v. cloning) • primer design: requires some baseline information about DNA sequencing of an Organism (or close relative) • prone to contamination: tiny amount of starting DNA needed, so require several negative controls - Can accidentally clone DNA from a different source; need to be cautious you are not amplifying your own DNA …Even PCR of tardigrade DNA! • “Water bears” (small and tough) survive at -273°C to 151 °C for 10 years without water • “Cheps” Sands (tall and spooky) molecular ecologist British Antarctic Survey More applications… • Species identification (i.e., illegal whale meat sales) • Chromosome walking (i.e., sequence unknown DNA regions, adjacent to known/well-characterized ones) - Essentially start at a particular place on a chromosome and extend outward; generating an entire chromosome sequence • Screen known mutations (e.g., allele-specific PCR) Following cloning or PCR • Restriction mapping: use a variety of Res to cut ‘target’ DNA, and electrophonically separate fragments • Establish number, order, and distance between cut sites • different alleles at a locus (DNA variation) uncovered by restriction fragment length polymorphism (RFLPs) Electrophoresis • Technique for separating DNA fragments according to differences in size using a gel to act as a viscous matrix as well as an electric charge to pass through this gel - Send electric current from negative –> positive end; DNA migrates towards positive end (Anode) - Bands with longer fragments will take more time to move through the gel - Bands with shorter fragments will take less time Medical Diagnosis • Screening disease-associated alleles via restriction fragment length polymorphism (RFLP) analysis • Sickle cell: single DNA substitution that leads to a different amino acid, and ß-globin protein - Homozygous: detrimental; heterozygous: effects show later • leads to change in (and loss of) RE recognitions sites… provides diagnostic RFLP banding pattern Sickle cell diagnosis using RFLP • MSTII cuts normal ß-globin 3x –> 2 short DNA fragments • MSTII cuts a single mutant ß-globin 2x –> 1 large fragment • Hets vs. homs (diploid genotype) Pharmacogenomics • Medication side-effects: common and sometimes fatal • medication effectiveness: variable, often works for only about 60% of the population • Pharmacogenomics: drug selection based on an individuals genetic makeup (rather than trial and error) - Individuals with same disease metabolize 6 MP differently - Differences due to genetic make-up (TPMT genotype) - Genetic assay allows customized treatment Complimentary DNA (cDNA) • cDNA: protein-coding DNA only… not non-coding DNA (including regulatory regions, e.g., promotors) 1. Extract mRNA from a particular tissue type, at a particular time (e.g., developmental stage) 2. Use Reverse Transcription PCR (RT-PCR) to convert mRNA back to DNA • The resulting pool of DNA represents only those - Complimentary DNA: protein-coding genes - The resulting pool of DNA represents only those __________. Reverse Transcription PCR • Mature mRNA = coding • Can use as template for reverse transcription • Oligo-DT primer anneals to poly-A tails (ds molecule) • Reverse transcriptase enzyme extends, makes complimentary DNA enzyme extends, makes complementary DNA • mRNA-DNA hybrid molecule created • RNase H enzyme nicks and digests mRNA strand • Remaining mRNA used DNA polymerase • ….dsDNA = expressed coding DNA (‘snap shot’) DNA Sequencing • Direct way to assay structure and organization of genes, and levels of variation within/among organisms • Determine the identity and order of nucleotides • Until recently, almost exclusively done via the “chain termination” (aka Sanger) sequencing method Chain Termination Sequencer • Raw data = set of colored peaks on a chromatogram • 1 peak per nucleotide (100bp fragment = 100 peaks) • Usually around 800bp reads • but…. Fairly slow and expensive Lecture 12 9/21 Learning Goals • Recall the steps involved in sequencing a genome • Understand the role of sequence alignment • Describe approaches used to annotate a genome • Give an example of comparative genomics DNA Sequencing • Direct way to assay structure and organization of genes, and levels of variation within/among organisms • Determine the identity and order of nucleotides - Been done for a number of years • Until recently, almost exclusively done via the “chain termination” (aka Sanger) sequencing method Chain Termination Sequencer (Sanger) • Raw data = set of colored peaks on a chromatogram - Colored peaks tell us which is A, T, C, or G in a nucleotide sequence • 1 peak per nucleotide (100bp fragment = 100 peaks) • Usually around 800bp reads (relatively long) • but…. Fairly slow and expensive (10 dollars each) “Next-generation” Sequencers • Illumina Genome Analyzer – 150-bp reads, fast and cheap • Roche/454 GS FLX – 400-bp reads, fast and cheap ** relevant to align and assemble nucleotide sequences** Ch. 18 Genomics and Bioinformatics New-ish fields • Genomics – structure and function of genomes (all of the DNA carried in an organism), often comparative • Proteomics – identify proteins present in a cell at a given time/location; modifications and interactions • Bioinformatics: filtering and analyses of very large genetic datasets; software development Genomics • The study of the complete genetic information of small creatures that live in the depths of the earth, who guard buried treasure Karyotype is highly variable Organism Diploid number (2n) African Wild Dog 78 Badger 32 Carp 104 Adders-Tongue 1262 (!) Cotton 52 Organismal “complexity” Not proportional to the number of chromosomes… …but what about genome size? (absolute number of base pairs) - Total amount of DNA within a haploid genome - Measured as the total number of base pairs (Kb – thousand, Mb – million, Gb – billion) Genome size also variable Organism Billion bp (1000 million) African Wild Dog 2.66 Snapping Shrimp 15.45 Carp 1.61 Poison Dart frog 8.70 Hugh-man 3.03 (human) How does number of chromosomes relate to absolute size of genome? - Size of African wild dog genome is quite larger than carps’ - Chromosomes are of different size (African Wild Dog’s are larger than carps) Sequencing a Genome • Genome – complete set of all the DNA in a cell; size is variable (usually: virus < prokaryote < eukaryote) • DNA sequencing – most methods have short “read lengths” (< 800-bp of continuous nucleotides) • Whole genome “shotgun” sequencing - Fragment the genome - Sequence all fragments - Assemble/order the pieces Genomic Libraries • large collection of DNA fragments (i.e., broken up whole genome), inserted/stored in vectors • Does not involve PCR… use RE digests of genomic DNA to make the fragments (varying size) • Try to get at least one copy of every sequence in the fewest number of fragments (use BAC or YAC clones) Shotgun Sequencing Technique • Fragment the genome (using REs), in slightly different ways • e.g., use a few different REs (but one at a time) • Gives different combos overlapping fragments • Sequence and align overlapping fragments (computationally) • Eventually, assemble whole chromosome sequence and repeat for all chromosomes Assembling “contigs” • Contig: a continuous stretch of DNA sequence (overlap) - Within just one gene - Across adjacent genes HP Computing (align, assemble) • de novo alignment and assembly – never done before, no baseline data • re-sequencing – already have a reference genome or “scaffold” Diploid Genomes • An individual can have a heterozygous genotype - Allele 1 v. Allele 2 - E.g., 1/1 (hom) vs. ½ (het) vs. 2/2 (hom) … so may need to align two variants of a given gene DNA Sequence Alignment • Scenario 1 – 2 reads do not start in same place - Align sequences to see a match-up - Mismatch between 2 nucleotides – point mutation/DNA sequence substitution - Q: are they otherwise identical? o A: NO Gene Pools • Population – gene pool can contain many alleles/allelic variants e.g., alleles 1, 2, 3, 4 … etc. … so may need to align >2 variants of a given gene - Look out for different kinds of polymorphisms DNA Sequence Alignment • Scenario 2 – many individuals; potentially many alleles - Align sequences to see a match-up - Q: but how many? o A: at least 5 Take multiple sequence alignment and count the number of different sequences that exist • Bioinformatics allows us to figure all of this quickly Annotating a Genome Interpretation and analysis of genome sequences relies on having names and locations assigned to genes Protein-coding genes have some hallmarks: - Have start codons (mRNA = “AUG”, DNA = “TAC”) o T pairs with A, A pairs with U, and C pairs with G - Open Reading Frame (no internal “stop” codons) - Have conserved upstream regulatory regions (e.g., TATA-box) o Binding sites – often have conserved genetic DNA sequences Can also compare to annotated sequences in databases: - BLAST searches to determine identity o Query data base with stretch of DNA or amino acid sequence and find the “best” match that exists in data base… can infer the likely chromosome or origin or function of the portion of DNA that is included in the search - Can query using DNA sequencing, or amino acid sequencing Public databases • National Center for Biotechnology Information (NCBI) - Contains DNA sequences, annotated DNA sequences from whole organisms and model organisms • Human chromosome maps, including known polymorphisms - Blue text – protein coding genes • Role of bioinformatics is not just to align and assemble existing short reads of DNA sequences to generate whole genomes, but also in comparison of whole genomes across individuals within same species or across different species • BLAST searches: can query NCBI database with an “unknown” stretch of DNA sequencing… find closest matches BLAST Searches • Query sequence –> stretch of sequence in which we generated and don’t know its chromosomal origin or its function; going to compare it to its closest match in the blast search - Best match highlighted in blue - FIG 18-3 • Mouse (subject) sequence already annotated, matching portion is an insulin receptor gene on Chromosome 8 • Rat (query) sequence was from an unknown genomic location, but identity and function can now be inferred - Can infer that the 280 base pair sequence from the rat probably originated from the same genomic location in the rat genome - Can also infer that the 280 rat sequence does not contain stop codons where they don’t belong if we expect this to be a protein coding sequence… o or if we were able to identify an AUG start codon or even an upstream regulatory region * stronger inference if rat sequence also has an ORF, etc. Comparative Genomics • covered so far - how a whole genome sequence is produced o shotgun sequencing – fragmenting, sequencing, and aligning and assembling o annotate genome by identifying through inference what the identity and function of DNA sequence is o once fully genome’d, compare it to the whole genome of a second, third, or fourth species • Comparing genomes of different organisms, some close relatives, others distantly-related - considerable similarity among organisms you would not have thought • Understand structure, function and expression of (mutant) genes involved in human diseases - 60% of genes from 300+ human diseases also in Drosophila - 60% of inherited dog diseases similar to those in humans o comparative genomics provides a way to identify useful model organisms that can help in experimental crosses that may inform us how to treat human genetic diseases • Important evolutionary insights from comparative genomics - direct comparison between genome sizes between different classes of organisms o prokaryotes, eukaryotes, viruses - Viruses – genome sizes small relatively; moderately strong correlation between genome size and number of protein coding genes - Prokaryotes – strong linear relationship between genome size and number of protein coding genes in the genome - Eukaryotes – diffuse relationship (but still correlated) but not that tight; some large genomes with relatively few protein coding genes (large portion of genome is comprised of non-coding DNA) Lecture 13 9/23 From Article: • Adaptive change evolved rapidly (w/in 6,000 years) • Predation rate increases for white mice in dark backgrounds and dark mice in lighter backgrounds - Suggests that phenotype is adaptive and depends on the context of the environment in which the mice lives • Single DNA sequence change in protein coding portion – amino acid replacement - Different protein variant that functions differently - How they figured out allelic variant with white coat colored is derived – A SNP is 1 nucleotide along a stretch of protein coding region… this is the only variant of focus that exists in natural populations…derived or inherited? o Used phylogenic approach o Derived because most mice in lineage is dark, only every now and then a white phenotype will arise o Tells us what the gene does • Describes details to controlled cross and gives indication of what the results mean - Dominance/recessiveness - 1 gene or more than 1 - gene is pleiotropic and RR is dark and CC is light so RC is intermediate - copy of normal allele and copy of mutant allele o look at figure 4 - proportion of chromosomes that carry the allelic variants - mosaic chromosomes – different phenotypes o 75 dominant phenotype and 25 recessive – standard Mendelian assumption does not hold in this case (varying phenotypes) not always equal fitness - Table 1 – shows results of the controlled cross focusing on the F2 generation and their phenotypes; genotypes have been sequenced o PVE is main column of interest because it shows the percentage of variancts explained • Figure 2 – basically what we get is 2 cell culture lines identical in every way except for which allelic variants of MC2R gene they have - Alleles were expressed - Figure shows how the 2 different cell lines perform with respect to generating melanin - MC1R has an activator which shows dark pigment and an antagonist which produces a lighter color o Lots of activating protein – normal allele produces lots of melanin while mutant allele does not produce as much Shows that DNA sequence mutation that alters amino acid is encoded to the protein produced –> respond differently in cell • The light coloration on the Gulf Coast is due to the MC1R allele while the light coloration of the mice on Atlantic coast is not due to the MC1R allele. This suggests that there are different ways to obtain that phenotype depending on the environment of the population - White population on Atlantic coast evolved differently – nothing to do with MC1R allele • Does evolutionary change proceed gradually through many small mutational steps or can adaptation occur via a few large leaps? • Does adaptation generally proceed through dominant or recessive mutations? Any of the above • Do beneficial mutations tend ot affect protein function, or ists spatial or temporal expression? • Are same genes and mutations responsible for similar traits in different poulations or species
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