BIOLOGY 305 1st Edition Lecture 17 Outline of Last Lecture I. Introduction to Methods Studying GenesII. Polymerase Chain ReactionIII. Analysis of DNA, RNA, and ProteinIV. Restriction AnalysisV. High Throughput SequencingVI. Vocabulary and Sample QuestionsOutline of Current Lecture I. Methods Continued (Identity Testing)II. Introduction to Replication and DiscoveryA. The Meselson-Stahl ExperimentIII. Mechanism of DNA ReplicationA. InitiationB. ElongationC. Termination IV. Vocabulary and Sample QuestionsCurrent LectureI. Methods Continued (Identity Testing)Short Tandem repeats – short sequence of DNA that is duplicated several times throughout an individual’s genome- it is hereditary, number of STRs depends on parents- the length becomes longer as it is repeated- can test many in one experimentHow it works:The STRs are colored differently and migrate on an electrophoresis gel at different rates based on number of nucleotides per STRCan use STRs to compare parent to child:Ex: does the dad in the figure below match the child?1) Yes, the child has a 19 STR from dad and 21 STR from mom2) No, this child lacks an STR in common with the suspect, thus this individual is not the fatherAt each locus, you have the same length as 5-20% of the rest of the population:#matches = 1/(.2^n), where n = number of loci testedmeans that: 1 in 1/3 trillion chance you will match someone else, but there’s not even that number of people on Earth, so there is high statistical power in this experimentII. Introduction to ReplicationRecall: Every daughter cell has to be the same as the parent cell so there must be a reliable mechanism for replication:- Must be extremely accurate (infrequent mutation) = able to replicate- Has to have ability to carry sufficient information- Must have the ability to mutate (for genetic diversity)The Meselson-Stahl Experiment:Using centrifugation to create a gradient by molecular weight of objects in the solution:E. Coli with only 15N (nitrogen isotope) DNA are transferred to a 14N medium and allowed to divide. Centrifuged DNA that is 15 N and 14N, 15N will migrate to the bottom, 14N to the top, DNA with a 15N strand hybridized to a 14N strand will be intermediate in the tubeSemiconservative Model: each molecule will have one old and one new strandIsolated DNA after the first cellular division:When the template separates, expect that every DNA molecule should have one strand from the parent (which is 15 N) and one new one:14NThen once again after the second division of replication, template separates:Expect that the new strand that forms is exactly like the template Result: heavy  intermediate  light and intermediate DNA bandsOther (disproven) models:Conservative Models1st round: One molecule is the entirely conserved “old” molecule and the other is all newly synthesized DNA2nd round: 25% old DNAResult: heavy  light and heavy band  light and heavy bandDispersive1st round: each strand will have a mix of old and new DNA2nd round: also all mixed, but not the same: see more new DNAResult: heavy  intermediate  slightly raised intermediateIII. Mechanism of ReplicationSemi-conservative model was well predicted by Watson and Crick: structure and functionBasically: strands are separated and then new strands are created in complement to the separated strands DNA is semi-conservative:Key: Direction of replication is always 5’  3’!Strand is unwound, direction of fork movement will be indicated and determines:The Leading strand: same direction as fork movement, continuous templateThe Lagging strand: synthesized by okazaki fragments (3’  5’ orientation)1) Exonuclease (5’  3’) removes primer2) Gaps are filled by DNA Polymerase (also 5’  3’)3) Gaps are sealed by ligaseBinding proteins protect this process and multiple okazaki fragments are often presentRecall:- Bacterial chromosomes are circular double stranded DNA with nucleoid proteins- Eukaryotic are linear, as in organized in to multiple linear chromosomesA. InitiationThink: What would happen if the chromosome is replicated twice: whole or just part of the chromosome?Helicase – separates DNA strands at two points at the ORI (Origin of replication)forms a replication bubble: ORI + two helicase, including two replication forksDNA Polymerase catalyzes DNA SynthesisCan’t just go to a section and replicate, need primers (recall PCR)always needs a 3’ end, this end is the part that growsB. ElongationCircular ReplicationGoes in both directions of the circular DNA from ORI to the termination regionReplicon – the length of DNA that is replicated following one initiation event at a single originthe genome of E. Coli has 1 repliconHas usually one ORI due to circular shape (eukaryotic cells have 2)Only for initiation (DNA alone does elongation) What happens at the end? The last okazaki fragment? See termination.Types of DNA Polymerase Note: DNA Polymerase does NOT produce Primers; this is done by RNA polymeraseNotice that exonuclease can work both ways: proofreading is 3’  5’!DNA Pol I does primer removal and filling of gapsDNA Pol II does DNA repairDNA Pol III doe DNA synthesisSummary of DNA Replication Machinery in Bacteria:The Replisome and Accessory Proteins:Starts with Helicase, DNA Pol III dimer works toward Helicase, RNA primer and primase works inopposite direction. The definition of the 3’ strand and the 5’ strand depend on which proteins are bound to itSample Problem:C. TerminationThe last okazaki fragment is shorter than the othersAfter removal of RNA Primer, DNA gets shorterThis problem is helped by Telomeres, which act as protective capsTelomeres (on the end of only Eukaryotic chromosomes) contain many repeats Telomerase is an enzyme that is elongates telomeres, a protein containing a piece of RNA, which is a reverse transcriptaseThe lagging strand will be longer in the next round of replication due to telomeraseStep 2 is the following after one round of replicationIf there is no telomerase, cells cannot divide. Often acts as a limitation for somatic cellsBut telomerase activity is activated in…Early embryonic cells, germ cells, stem cells, and many cancer cellsIV. Vocabulary and Sample Questions:Semi-conservative – each DNA double helix contains one old and one new strandHelicase – enzyme responsible for unwinding the DNA double helix, resulting in a replication bubbleReplication fork – ½ of a replication bubbleDNA polymerase – enzyme that