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UGA BIOL 1107 - Test 3 Study Guide

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DNA Structure and Replication Make a diagram of a DNA molecule that accurately depicts the relationship and polarity of each DNA strand in the molecule 5 ATAGCCGCC TAATTAATTGA 3 3 TATCGGCGG ATTAATTAACT 5 5 contains a Phosphate group which is acid whereas the 3 end contains a hydroxyl group which is basic Describe how cells replicate their DNA and predict how replication would be affected if one of the major enzymes involved was missing from the cell Helicase breaks the Hydrogen bonds between strands starting in an area rich in A T forming bubbles which combine to form a replication fork Strands would be unable to separate RNA primase binds to the DNA and elongates the strands from the 3 5 direction The leading strand is continuous whereas the lagging strand forms in segments the strand would not be able to be copied and elongated DNA Polymerase removes primase and with Ligase reads and combines the Okazaki fragments forming a complete elongated lagging strand the lagging strand would remain in fragments In termination DNA polymerase reaches the ends of the strands The two new strands are semiconservative The telomeres are not replicated The template strand would not be able to split into daughter strands Repair Compare and contrast DNA replication and the Polymerase Chain Reaction PCR Both Double stranded DNA is unwound Each strand acts as a template for the synthesis of a new strand End product is double stranded DNA Reaction is performed by a DNA polymerase dNTPs are the building blocks DNA Replication Naturally occurring process Generates a full length new DNA molecule Lower temperature PCR Lab reaction Specific region defined by the primers in copied Higher temperature Explain how gel electrophoresis is used to analyze DNA Gel electrophoresis is used to separate out proteins or DNA by size Both proteins and DNA have an overall negative charge so an electric field is used to pull the proteins DNA towards the positive electric terminal The gel matrix they are being pulled through is somewhat thick so small pieces will travel further than bigger pieces Interpret results from gel electrophoresis A typical sample contains plasmids as well as contaminating RNA and chromosomal DNA Most chromosomal DNA is sheared into large linear pieces during cell lysis and they appear within the first third of the gel Because the fragments are of different sizes this band is rather diffuse Large DNA and protein DNA complexes remain at or near the sample wells Ribosomal RNA the major RNA in cells migrates very quickly and usually co migrates with bromophenol blue tracking dye The remaining bands are generally plasmid DNA Covalently closed circular plasmids also known as supercoiled plasmids migrates faster than the linear form which in turn migrates faster than the opencircular form also known as nicked plasmids Circular plasmids may also dimerize The analysis of whole plasmid migration is not trivial Fortunately complete digestion of the plasmids into linear forms make for easier analysis To check for incomplete digestion it is important to include a lane of undigested DNA in a restriction run EtBr staining of gels has a sensitivity of 10 100ng of DNA For routine checking of DNA running 1 mg of DNA is more than enough To get better resolution thinner bands run 200 300ng Overloading the lane will result in extensive streaking For small fragments 300 bp and below it may be necessary to load more DNA as their small size means that they intercalate less EtBr In addition EtBr runs in the opposite direction as DNA and the lower part of the gel is usually depleted of EtBr after a run Estimation of the molecular weights of DNA is usually made by comparing bands to DNA markers For a more accurate calculation the distance of migration Rm of the markers is plotted against the log of the molecular weight MW The molecular weight of the unknown sample is extrapolated from the plot The Rm versus log MW plot is a curve and molecular weights obtained from the top or bottom of the graph will not be accurate Explain how DNA can vary from one individual to the next and how these differences can be used to create a DNA fingerprint The sequence of nucleotides is different thus changing the amino acids produced thus changing the proteins final structure thus creating different final whole organisms Complementary Each of the two strands of DNA corresponds to each other based on base pairs Antiparallel Orientated in opposite directions Single Stranded Binding Proteins Bind to unpaired DNA strands of DNA are being unwound Ligase Joins Okazaki fragments together DNA fingerprinting the analysis of DNA from samples of body tissues or fluids in order to identify individuals Template Provides pattern of base pairs to be copied by RNA for transcription Daughter Strands Newly synthesized strands from template strand resulting from the fork Primer Short stretch of RNA with free 3 end bound by complementary base pairing to the template strand and elongated with DNA nucleotides during DNA replication 5 End 3 End Way that DNA is orientated and synthesized DNA Polymerase Catalyze synthesis of new DNA by adding nucleotides to pre existing chain Semi Conservative produces two copies such that each contains one of the original strands and one new strand Replication Fork Y shaped region where parental strands of DNA are being unwound Bubble Connect and unwind to form a replication fork Gel Electrophoresis Technique for separating nucleic acids or proteins on the basis of their size and electrical charge both of which affect their rate of movement through an electric field in a gel made of agarose or another polymer Molecular Weight Marker protein ladder DNA ladder or RNA ladder is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis using the principle that molecular weight is inversely proportional to migration rate through a gel matrix Helicase Enzymes that untwist the double helixes at forks separate the two parent strands and make them available as templates Gyrase enzyme that relieves strain while double strand DNA is being unwound by helicase Primase An enzyme that joins RNA nucleotides to make a primer during DNA replication using the parental DNA strand as a template Leading Strands made by continuous addition of nucleotides by DNA polymerase Lagging Strands Elongated away from fork synthesized discontinuously each segment is an Okazaki fragment PCR used to amplify a


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