1Lecture 8: Molecular Markers and mappingRead 391-408Fig. 11.3; 11.4; 11.6-8; 11.10-15Table 11.1Next generation DNA sequencingDNA polymorphism: the basis of molecular markersMethods of detection and application (diagnosis,finger-printing)2Personal Genome Project (PGP) Goal: sequencing full genome of individuals at $1000Human Genome Project (HGP) (total 3 billion $) :Motivated 100X reduction in cost (10 $ per base to 10 base per 1$.)Personal Genome Project(PGP: has motivated thedevelopment of Ultra-Low-Cost-Sequencing (ULCS).3ULCS under developmentMicroelectrophoretic sequencingHybridization sequencingCycle-array sequencing on amplified molecules (SBS)Pyrosequencing (454)Reversible terminator sequencing (Solexa)Restriction digestion and ligation (MPSS-Lynx)Fluorescent In Situ Sequencing (FISSEQ)(Multiplex in space and time; Avoidance of bacterial clones)Nanopore sequencing4PyrosequencingDetects extension through luciferase-based real time monitoring of pyrophosphaterelease.5Step 1: PCR amplified ssDNA templates is hybridized to a sequencing primer and incubated with theenzymes: DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine5´ phosphosulfate (APS) and luciferin.Step 2: The addition of one of the four deoxynucleotide triphosphates (dNTPs) initiates the secondstep. DNA polymerase will catalyze the incorporation of dNTP onto the template if it iscomplementary. It is important to note that if there is an incorporation there will be a release ofpyrophosphate (PPi) equivalent to the amount of dNTP incorporated.Step 3: ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin thatgenerates visible light in amounts that are proportional to the amount of ATP. The light produced inthe luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and this canbe analyzed in a program. Each light signal is proportional to the number of nucleotidesincorporated.Step 4: To continue the sequencing the degrading of nucleotides is essential. Apyrase is anucleotide degrading enzyme and does in this step clean the solution from all dNTP.Step 5: New nucleotides can be added and a new cycle can start.Pyrosequencing invented by Mostafa Ronaghi, furthered by BiotageRonaghi, M., Uhlen, M., and Nyren, P. 1998. Science 281: 363. A sequencing method based onreal-time pyrophosphate.67Megaclone™ technology(invented by S. Brenner and patented by Lynx Therapeutics) Megaclone™ transforms a sample containing millions of DNA molecules intoone made up of millions of micro-beads, each of which carries approximately100,000 copies of one of the DNA molecules in the sample.http://mpss.udel.edu/tutorial/flash/megaclone.swf8Reversible terminator sequencing9Nanopore sequencingSingle stranded polynucleotides can only pass single file through a hemolysinnanopore. The presence of the polynucleotide in the nanopore is detectedas a transient blockade of the baseline ionic current. PA-pico-Ampere10You tube:Pyrosequencingnanopore sequencing“ 23 and me”Website: Personal Genome ProjectGoogle11Four classes of DNA polymorphisms12Single nucleotide polymorphism (SNP)• Single base-pair substitutions• Arise by mutagenic chemicals or mistakes inreplication• Biallelic – only two alleles• Ratio of alleles ranges from 1:100 to 50:50• About 10 million human SNPs identified (23 and meassays 550,00 SNPs) (Chimpanzee and human have35 million SNPs and 5 million indels)• Most occur at anonymous loci• Mutation rate of 1 X 10-9 per locus per generation• Very few are thus new mutation in the species• Useful as DNA markers13Microsatellites• 1 every 30,000 bp• Repeated units 2 – 5 bpin length• Mutate by replicationerror• Mutation rate of 10-3per locus per gamete• Useful as highlypolymorphic DNAmarkersFig. 11.314Minisatellites• Repeating units20-100 bp long• Total length of0.5 – 20 kb• 1 per 100,000bp, or about30,000 in wholegenomeFig. 11.415Deletions, duplications, and insertions• Expand or contract the length ofnonrepetitive DNA• Small deletions and duplications arise byunequal crossing over• Small insertions can also be caused bytransposable elements• Much less common than otherpolymorphisms16LerColHindIIIHindIIIHindIIIHindIIIHindIII2 kb5.7 kbLer Col Het1kb ladder521Probe:RFLP: Restriction Fragment Length PolymorphismHindIII digestionElectrophoresisSouthern blottingHybridization with the probeautoradiography17• SNP detection usingsouthern blots– Restriction fragment lengthpolymorphisms (RFLPs)are size changes infragments due to the loss orgain of a restriction siteFig. 11.618Example of RFLPRestriction digestionGEEthedium bromide stainBlottingHybridizationX-ray radiography19CAPS (Cleaved Amplified Polymorphic Sequences) ie. RFLP detection by PCR LerColHindIIIHindIIIHindIIIHindIIIHindIII2 kb5.7 kbLer Col Het1kb ladder521PCRHindIII digestionGEL C M20CAPS• Must have sequence oneither side ofpolymorphism– Amplify fragment– Expose to restrictionenzyme– Gel electrophoresis• e.g., sickle-cellgenotyping with a PCRbased protocolFig.
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