DNA Replication Sequencing PDF

Applications derived from the principles of DNA Replication: Sequencing (using replication principles) All course content is copyrighted and remains the intellectual property of...

Applications derived from the principles of DNA Replication: Sequencing (using replication principles) All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Lecture Overview Sanger Sequencing Pyrosequencing Illumina sequencing All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Sanger sequencing Sequencing approach based on PCR Requires a forward primer DNA template DNA polymerase Normal dNTPs ~ 1 kbp fragment reads at a maximum Sequencing by (chain) termination Implements dideoxynucleoside triphosphates Lack of free 3’ OH group prevents further extension All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Sanger sequencing - initial process Run four sequencing reactions simultaneously Each reaction contains all four dNTPs and only one ddNTP When base complementary to ddNTP is encountered, either dNTP or ddNTP is incorporated = each reaction have a collection of fragments of different lengths https://www.researchgate.net/publication/234248746_Simulation_of_polymer_translocation_th rough_small_channels_A_molecular_dynamics_study_and_a_new_Monte_Carlo_approach All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Sanger sequencing - initial process Sequencing reactions run on a polyacrylamide gel Fragment lengths separated for each reaction Can read the sequence from gel Theophilus, B.D.M. (2000). Preparation and Analysis of DNA Sequencing Gels. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:481 All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Sanger sequencing - improvements Started using ddNTPs with fluorochromes attached Single reaction with 4 dNTPs and 4 differentially fluorochrome labeled ddNTPs Each fragment fluoresces based on ddNTP that terminates the chain Separate the fragments through capillary gel electrophoresis Theophilus, B.D.M. (2000). Preparation and Analysis of DNA Sequencing Gels. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:481 All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Uses of Sanger sequencing Used to sequence - Small regions (genes or to fill in gaps left by “next gen” sequencing) Confirm cloning constructs (e.g., detect errors in a PCR product) Was initially the only type of sequencing available - Used to sequence entire genomes in ~ 1 kbp fragments Need to know a starting sequence to design a primer. required short genomic fragments incorporated into plasmids All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Next generation sequencing (NGS) – “Sequencing by synthesis” Works on the principle of detecting which nucleotide is incorporated during polymerization by DNA polymerase Massive parallel sequencing DNA sheared into smaller pieces that are sequenced and need to be re- assembled Need free 3’ OH group and template Different platforms differ in chemistry https://microbenotes.com/next-generation-sequencing-ngs/ All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Pyrosequencing https://microbenotes.com/pyrosequencing/ 1st NGS platform that is mostly phased out DNA fragments attached to solid surface (microarray) Microarray flooded with a particular dNTP at a time Pyrophosphate release through dNTP incorporation sets cascade into motion to generate light Light intensity is an indication of the number of dNTPs incorporated All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Pyrosequencing – light detection Requires: 1. DNA polymerase (No 3´→ 5´exo) 1. DNA polymerase incorporates a dNTPs (releases PPi) 2. dNTPs 2. Sulfurylase converts PPi and APS → ATP 3. adenosine 5´ phosphosulfate (APS) 3. Luciferase converts Luciferin to oxyluciferin 4. Sulfurylase 4. Light is detected by a camera and analyzed by computer 5. Luciferase software 6. Luciferin 5. Apyrase degrades the unincorporated nucleotides and ATP 7. Sequencing primer Cycle continues with next dNTP 8. Apyrase All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Pyrosequencing – light detection https://microbenotes.com/pyrosequencing/ All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Illumina sequencing Steps of Illumina sequencing: 1) Adaptors are added to your template DNA 2) Bridge amplification of your template 3) Sequencing by synthesis 4) Analysis All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Illumina sequencing Step 1: Adaptors are added to your template DNA DNA gets chopped up dsDNA adapters (short, known DNA sequences) are added via ligation to flank DNA Contain primer binding sites for amplification and sequencing Modified genomic DNA is melted into ssDNA and affixed to chip surface at one end All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Illumina sequencing Step 2: Bridge amplification of template Hundreds of oligonucleotides complementary to adaptors are attached to surface of chip Base pairs with free primer end and serves as primer with free 3’ OH end DNA Polymerase can generate complementary strand Generated dsDNA denatured and process repeated to form “clusters” of sequences on surface All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Illumina sequencing Step 3: Sequencing by synthesis Primers and dNTPs with fluorescent tags floods surface of chip Have a 3' blocker that prevent continued polymerization Single nucleotide added during each round of synthesis After each round of synthesis, a camera takes a picture of the chip Computer interprets fluorescent signal to determine which dNTP was incorporated This is recorded for every spot on the chip All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Illumina sequencing Step 3: Sequencing by synthesis Non-incorporated molecules washed away in between each round of synthesis Chemical deblocking agent removes blocking moiety and fluorescent dye Process repeated until full DNA molecule sequenced Can sequence thousands of molecules simultaneously (parallel sequencing) All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Illumina sequencing Step 4: Analysis Raw sequence data of short fragments recovered from sequencing Sequence reads need to be assembled through stacking overlapping regions Scaffolding process of stacking overlaps repeated until contigs (pieces of contiguous sequence) are assembled Individual errors that could have been introduced gets diluted out by the scale of the sequencing All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.  Applications of sequencing 1. Genome sequencing of a single organism 2. Metagenomic sequencing of all DNA in a community 3. Sequencing of all RNA transcripts (how genome data relates to actual expression) All course content is copyrighted and remains the intellectual property of Dr. Marike Palmer. Please do not distribute this material without the express permission of Dr. Marike Palmer.