Genetics Study Guide Test 2
Genetics Study Guide Test 2 BIL250
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This 9 page Study Guide was uploaded by De Vs on Monday February 23, 2015. The Study Guide belongs to BIL250 at University of Miami taught by Dr. McCracken in Fall. Since its upload, it has received 324 views. For similar materials see Genetics in Biology at University of Miami.
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Date Created: 02/23/15
77 Explain Barbara McClintock39s ndings of autonomous and nonautonomous transposable elements in corn discovered mobile genetic elements plant transposons have ITR sequences generate short repeats at target sites activaterepress target genes cause chromosome mutations and disrupt genes autonomous elements transpose themselves and have a transposition gene non autonomous elements cant and rely on another Tn she found that purple spots on white corn is from transposable elements c allele is formed from a non autonomous transposon Ds inserted into the C gen The ds transposon is controlled by an autonomous transposon Ac Ac activates D5 D5 is a rearranged version of Ac if reversion of c to C occurs in a cell it makes a purple spotted corn normal purple corn is C 78 How can nonreplicative transposition result in a net increase in transposable elements if it occurs during chromosome replication transposition to an unreplicated recipient site results in a net increase in the number of transposons when the region of the chrom containing the Ac element is replicated 79 What makes a retrotransposon different from a transposon retrotransposons encode reverse transcriptase and make DNA copies of RNA transcripts transposons encode proteins that move DNA directly to a new position or replicate DNA and integrate replicated DNA elsewhere in the genome DNAD RNA l DNA human retrotransposons are LINES autonomous ad SINES nonautonomous 81 What 4 important steps are required to clone DNA and create a recombinant library isolate DNA use SDS to break up cell membrane NaCl to lyse cells and bind DNA strands proteinase K to remove chromatin EtOH to precipitate and wash DNA and water cut DNA with restriction enzymes restriction enzymes recognize restriction sites in DNA and cleave by hydrolyzing phosphodiester bond cut occurs btwn 339 carbon of one nucleotide and the phosphate of the next ligate DNA into a cloning vector to make recombinant DNA molecule if the DNA to be cloned and the vector are cleaved by the same restriction enzyme the two different molecuels can bease pair together transform recombinant DNA into a host that will replicate 82 Identify the advantages and disadvantages of three different types of cloning vectors YAC BAC plasmid fosmid cosmid phage shuttle plasmids can replicate autonomously unique restriction site such that an enzyme cuts in only one place selectable marker to distinguish plasmids that contain inserts o Disadvantage is that vectors can reanneal with no insert recircularization it can only clone a gene up to 10kb 0 only replicate in one host phage vectors allow larger inserts and the ability to place more clones on a plate making it easier to grow large numbers of clones in a small space they kill their bacterial host when replicating the DNA insert BAC can clone large pieces of DNA they do not undergo rearrangements in the host can be used in the blue white colony screening method BUT AT rich DNA fragments do not clone well and some sequences are toxic to e coli and unclonable YAC can clone very large sequences but some vectors accept two or more inserts and the insert DNA is often deleted or modi ed by the host cell it39s a shuttle vector so it can be introduced into two or more diff host organisms shuttle vectors can be moved from one host species to another 83 What features of pUC19 make it a functional cloning vector Which of these features are contained in the polylinker region features that make it a functional cloning vector ori selectable marker unique polylinker sites polylinker region multiple cloning site region containing several unique restriction sites where foreign DNA can be inserted high copy number very active ori selectable marker is amp so only e coli with the plasmid will grow on medium polylinker occurs in the lacZ gene adding xgal turns blue in presence of b galactosidase meaning no DNA has been inserted white means DNA was inserted when DNA is cloned into the polylinker site the lacZ is disrupted and no function b galactosidase can be produced 84 If you were making a genomic DNA library why might you choose to use a partial digestion instead of a complete digestion genomic library collection of clones that contains at least one copy of every DNA sequence in an organism39s genome there are functional limitations with the library after cloning Avg size of the fragment produced by digestion of eukaryotic DNA with restriction enzymes is small and most genes are larger than 4kb there is a loss of info they cut in the wrong place etc so we need to break the DNA into fragments that are the correct size for the vector and overlap each other complete digestion makes many non overlapping DNA clones it digests at all relevant restriction sites partial digestion only a random portion of the restriction sites is cut by limiting the amount of enzyme used or the time of incubation DNA fragments can be cloned directly since they have sticlq ends 85 What makes a cDNA library different from a genomic DNA library Identify the steps used to create a cDNA library to illustrate your answer cDNA library clones of DNA copies made from mRNA isolated from cells reverse transcriptase RNA dependent DNA Pol synthesizes DNA from RNA template cDNA library shows genes that are transcribed into proteins it doesn39t have all introns and crap isolate mrna use reverse transcriptase to synthesize DNA and then clone the cDNA Steps 0 reverse transcriptase used to make a single strand DNA copy of the mRNA then RNase H DNA pol I and ligase make the double stranded DNA copy called cDNA cDNA has blunt ends so restriction site linkers adapters added to make sticky use T4 DNA ligase and blunt end ligation to add restriction site linkers to each end digest the linkers with the same restriction enzyme used to cleave the vector mix cDAn with cut vector DNA in presence of DNA ligase transform into host for cloning if cDNA has same restriction site as linkers cDNA will be cloned in pieces O 0000 86 Describe the Southern Blot setup Why would a geneticist want to use one of these Invented by Edward Southern used to probe DNA to determine the arrangement of restriction sites in the genome used for comparing homologous genes in dff species analyzing introns screening individuals for restriction endonuclease site differences associated with disease genes DNA on the gel is denatured using alkaline solution gel placed on blotting paper and a hybridization membrane goes over the gel stack paper towels and weight on top of membrane buffer solution is wicked up by blotting paper to the paper towels passing thru the gel and transferring DNA to membrane lter fragments on the lter are arranged the same as on the gel x DNA in place on membrane by baking in hybridization oven saturate the membrane with a homologous probe that binds speci c sequence and expose to x ray lm diff fragments of diff sizes separated uing gel electrophoresis to help determine location of restriction sites in the genome for sequencing 87 What are the PCR reaction contents and what thermalcycling steps are required PCR ampli es a small region of DNA target sequence to make unlimited complies without cloning the region thermal cycler is a machine that makes temp changes for speci c periods of time Steps 0 start with a DNA template and add two primers that are complementary to the ends of the target sequence 0 denature the DNA at 94 C into single strands 0 cool btwn 3765 C so primers anneal to complementary single stranded sequences at the ends on opposite strands o extend primers at 7075 C using heat resistant Taq polymerase 0 Repeat denaturing annealing and extension to make billions of copies 0 nal extension extend primers at 7075 C again to allow incomplete extension products to extend completely 0 cool to 4 C and store 89 Contrast manual dideoxy and automated dyeterminator DNA sequencing A clone from the library must be sequenced to determine the nucleotide sequence and the distribution of restriction sites the info tells us the properties of a protein a gene codes for and we can compare organisms Fredrick Sanger made dideoxy sequencing that uses synthesis of a new DNA chain on a cloned template DNA strand Synthesis of new strands is stopped by ddNTPS so they stop at all possible nucleotide positions this allows the complete DNA sequences to be determined dideoxy steps 0 DNA template denatured into single strands 0 single primer annealed to template DNA and extended with DNA pol 0 four rxns set up each with DNA template primer annealed to template DNA DNA pol and dNTPS a different labeled ddNTP is added to each tube these have a 339H instead of a 339OH and compete in the rxn with the normal dNTPS whenever a ddNTP is in the chain the syntheses stops each tube makes a population of DNA that terminate at diff positions each is electrophoresed on polyacrylamide gel which are faster pattern of bands is visualized using xray lm or automated sequencer location of bands indicates size of the fragment terminating with a certain labeled ddNTP Automated steps 0 ddNTPs are labeled with uorescent dyes o 4 dyes are combined that uoresce at diff wavelengths in one rxn tube and electrophores in one lane on a capillary thinner than gel and faster 0 UV laser detects dyes and reads sequence 0 sequence is displayed in a chromatogram with colored peaks that correspond to position of each nucleotide in the sequence O 0000 101 Describe the procedure used to quotshotgunquot sequence and reassemble a genome overview whole genome is broken into partially overlapping fragments each fragment is cloned and sequenced and and then assembled into larger sequences using computer algorithms gaps present in the sequences are lled by another sequencing process called nishing Steps 0 Random partially overlapping small fragments 2kb bp are generated by mechanical shearing o lsolate on agarose purify and clone into standard plasmid vectors 0 Sequence 500 bp from each end of each 2 kb insert 0 Sequence from the middle 1000 bp of each insert is obtained from overlapping clones using computer 91 What characteristics of microsatellites make them ideal for DNA ngerprinting they are polymorphic they have a single locus occur in only one location in the genome they are common throughout genomes of most organisms they are neutral STRs are 26bp DNA sequences that are tandemly repeated they are polymorphic so are good for forensics PCR is good for amplifying since its short the location on the chrom locus for each gene is mapped polymorphism is one of two or more alternate forms alleles of a chromosomal locus that differ in nucleotide sequence or have variable numbers of tandem repeats indels polymorphisms include SNPs STRs microsatellites VNTRs 92 How would you locate a proteincoding gene if the protein already is identi ed How would you locate the gene if the protein is unknown 0 rst one make cDNA and put it in a vector probe with antibodies that bind the gene product isolate and sequence positive clones if its unknown you use walking orjumping more dif cult identify a marker positional cloning and walking to hone in on gene and sequence 102 What is quotproteomicsquot and why is it important to study of disease proteome complete set of expressed proteins in a cell at a particular time prometomics is the cataloging and analysis of those proteins to determine when a protein is expressed how much is made and what other proteins it can interact with 0 most gene products involved in disease are composed of protein diseases are caused by problems with proteins if you understand where it comes from you can gure out the gene 93 What is the difference between quotchromosome walkingquot and quotchromosome jumpingquot How were these techniques used to isolate the cystic brosis gene 0 for mapping large sequences you do it before shotgun you use markers piece of sequence you walk through the chrom sequence once piece and get 700 letters that lets you map the next 700 0 cut out one sequence and 0 some SNPs affect restriction sites and can be detected using the restriction enzyme for the site the different patterns of restriction sites makes RFLPs restriction fragment length polymorphisms which are restriction enzyme generated fragments of different length 0 DNA polymorphisms that are useful for geneic mapping are DNA markers 0 a recurring RFLP showed association to cystic brosis trait CF gene was localized to chrom 7 using labeled RFLP probe and in situ hybridization and two more RFLPs were found to be linked section of chrom 7 was cut cloned and mapping by walking and jumping 0 walking 0 end of a cloned sequence is used as a probe to nd adjacent overlapping fragments in a genomic library 0 clones that overlap are mapped with RFLPs to determine the extent of overlap 0 new labeled probe designed for the second clone is used to screen the library againand this is repeated 0 jumping 0 partial restriction digestion used to cut large section of chrom DNA into large overlapping fragments o circularize fragements with DNA ligase to bring ends of DNAs that previously were distant closer together 0 cut the circles with restriction enzyme again to release the junction region ends now inverted 0 clone junction regions form a jumping library 0 subclone a small fragment of DNA and use as a probe to nd the next junction fragment occurring in the library same techniaque as walking 0 jumping reaches the target gene faster than walking 0 111 What are Mendel39s principles and why are they important principle of uniformity F1 offspring from monohybrid cross of true breeding strains resemble only one of the parents Principle of segregation recessive characters masked in the F1 progeny of two true breeding strains reappear is a speci c proportion of the F2 progeny inheritance is particulate not blending principle of independent assortment alleles for different traits assort independently of one another genes on diff chroms behave independently in gamete production testcross used to nd an unknown genotype you cross the indiv with a homozygous recessive when you don39t know anything about its genes monohybrid for one trait and Dihybrid for two traits rst law two members of an allele segregate from each other in the formation of gametes half the gametes carry one allele and the other half carry the other 0 second law pairs of alleles for genes on diff chroms segregate independently in the formation of gametes gene region of DNA that controls a speci c trait allele one of two or more alternative forms of a gene for a single trait that come from mutation and are found at the same place on a chrom R or r 121 Compare and contrast Y chromosome and X chromosomeautobalance mechanisms of genotypic sex determination in drosophila and c elegans the Y chromosome has no effect on sex determination sex is determined by the ratio of X to autosomes in mammals XY is male and XX is female 131 What is the molecular basis for complete incomplete and codominance Complete dominant aee creates fu phenotype by one of two methods 0 half the amount of gene product produced by homozygote is suf cient OR if expression of dominant allele in heterozygote is up regulated to match the homozygote the dominant allele stops anything else from getting expressed o haplosuf cient condition where one copy of a gene in a diploid organism is suf cient to give a normal phenotype incomplete one allele is not completely dominant to another phenotype of the heterozygote is intermediate btwn the phenotypes of homozygotes for each aee red and white makes pink 0 recessive allele is not expressed in heterozygote co dominance aees are codominant to one another phenotype of the heterozygote includes the phenotype of both homozygotes 0 both alleles are expressed equally resulting in a combined phenotype 132 How does recessive epistasis in uences coat color in rodents epistasis interaction btwn alleles of two or more genes to control a single phenotype one gene masks the phenotypic expression of another gene 0 genes mask other genes and affect expression F2 is 934 caused by recessive alleles aa masks the effect of B at another locus 141 Explain how Harriet Creighton39s and Barbara McClintock39s studies of corn in 1931 provided the rst convincing experimental evidence for Morgan39s hypothesis of recombination and chromosomal exchange 0 142 Describe the formation of the Holliday intermediate and explain how cleavage of this molecular structure can result in parental and recombinant allele combinations 0 143 Use the twopoint recombination frequency data from question 146 at the back of your book to map the genes a b c d and e 0 144 Order tetrad experiments indicate that crossing over occurs at the 4chromatid stage prophase l of meiosis What is the alternative What features of the ordered tetrad experiments make them ideally informative in this respect 145 What is the process of gene conversion and why is it important 146 Illustrate and example of mitotic recombination PCR vs Cloning PCR is faster than cloning but PCR requires using speci c primers and there must be sequence info available for the DN to be ampli ed the length of the DN that can be ampli ed by PCR is limited by the enzyme and coniditons to about 40kb taq polymerase has no proofreading activity transposable eIement mobiIe genetic elements of a chromosome that have the capacity to move from one location to another in the genome prokaryotes transpose tofrom new positions on the same chromosomes plasmid or phage chrom insertion sequences simplest in bacteria and plasmids encode transposase for mobilization and insertion the ends have inverted terminal repeats can disrupt coding sequencesregulatory regions aIter expression of nearby genes cause deletions and inversions cause crossing over 0 original copy stays in place and new copy inserts randomly transposition requires transposase which is coded by the IS eIement transposition initiates when transposase recognizes ITRs at target site staggered cuts are made in DNA at target site by transposase and DNA poigase ll in gaps transposon o composite carry genes that have inverted IS elements on both sides 0 noncomposite carry genes that don39t terminate with IS elements they are non IS eIement repeats eukaryotes transpose tofrom new position on the same OR a diff chrom they can leave a copy behind or just move to the new site yeast ty transposable element is a retrotransposon reverse transcriptase uses an RNA template to produce a DNA copy cDNA integrates at new chromosomal site 2 types of transposable elements 1 Encode proteins that 1 move DNA directly to a new position or 2 replicate DNA and integrate replicated DNA elsewhere in the genome 2 Retrotransposons encode reverse transcriptase and make DNA copies of RNA transcripts new DNA copies integrate at different sites eukaryotes only DNA Cloning Steps DNA microarray glass sides spotted with thousands of different DNA probes DNA probe molecule in an experiment used to determine if a complementary DNA or RNA target molecule is present gene function can be assigned experimentally by knocking out a gene or knowcking down ints expression and seeing the phenotypes that result different methods are used such as replaceing the normal chromosomal copy of the gene with a disrupted copy and inactiviating the gene by inserting a transposon into it bacteria if the eletion is successful then the gene got replaced with the selectable marker and permanently changed the chromosome BUT expression can be knocked down by RNA interference where a small regulatory RNA targets a speci c mRNA for degradation there I sno permanent change but revents translation of the mran of a targeted gene for as long as the small regulatory RNA molecule is present genetic engineering using recombinant DNA technology to manipulate genes for genetic analysis or develop products PCR usese oligonucleotide preimeres to amplify a speci c segment of DNA aplications in research generaties DNA for cloning or sequencing ampli es DNA for genetic defects ampli es for DNA ngerprinting if cDNA is used as a template for PCR mRNAS can be detected and quan ed
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