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First Sequencing Methods

First Sequencing Methods. Sebastian Beuchelt BIOL 446. Dr. Adema , Dr. Natvig 23 Sep 2019. Big Ideas. DNA — hereditary material written in nucleotides. Sequencing — "reading" Reverse genetics — modern basis for knowledge of genome organization.

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First Sequencing Methods

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  1. First Sequencing Methods Sebastian Beuchelt BIOL 446 • Dr. Adema, Dr. Natvig • 23 Sep 2019

  2. Big Ideas • DNA — hereditary material written in nucleotides. • Sequencing — "reading" • Reverse genetics — modern basis for knowledge of genome organization. • Earliest form of nucleotide sequencing — RNA sequencing

  3. Why Sequence? • Altered or non-functional protein. • Harmful, neutral, or positive effects. • Understanding genetic conditions. • Treatments.

  4. Sequencing: DNA vs. Gene vs. Genome

  5. Sequencing: DNA vs. Gene vs. Genome DNA Gene Gene — distinct portion of the DNA that codes for a protein or RNA. Gene sequencing — determination of the order of the nucleotides in an individual gene. • DNA — chain of nucleic acid. • A, T, C, and G. • DNA sequencing — determination of the order of the nucleotides in an individual’s DNA. ***not all DNA sequences are genes (i.e. coding regions, promoters, tandem repeats, introns, etc.) depending on organism and source of the DNA sample.

  6. Sequencing: DNA vs. Gene vs. Genome Genome • Genome — complete set of genes. • Genome sequencing — determination of the order of the nucleotides in an individual’s entire genetic material. • Genomics — sequencing and determining function of proteins, genes, and metabolic pathways in an organism.

  7. First Sequencing Methods

  8. Lots of Ways of Sequencing • Dye-terminator sequencing • Capillary electrophoresisº • Pyrosequencing*º • Sequencing by ligation • Sequencing by hybridization* • Next generation sequencing*º • Early RNA sequencing • Wandering spot analysis • Chemical cleavage method • Chain termination method* • Cycle sequencing* • High-throughput sequencing*º * still used today º techniques, not strictly sequencing methods

  9. Early RNA Sequencing • Insulin • First complete protein sequeced • Fredrick Sanger (1955) • Bacteriophage MS2 ((+)ssRNA) • First complete gene/genome sequenced • Walter Fiers et al. (1972) • Read RNA nucleotides by matching with amino acid sequences • RNA = smaller than DNA • Basis for RNA-sequencing

  10. Early RNA Sequencing PROS: First form of sequencing. Revolutionary ideas. CONS: Poor results. Lots of work. Difficulty scaling to genomes. Uses RNA and amino acids rather than DNA.

  11. Wandering Spot Analysis • Walter Maxam and Allan Gilbert (1973) • lac Repressor • First DNA sequenced • reported sequence of a “whopping” 24 base pairs • 5′--T G G A A T T G T G A G C G G A T A A C A A T T--3′ • 3′--A C C T T A A C A C T C G C C T A T T G T T A A--5′

  12. Wandering Spot Analysis • dsDNA fragments denatured into ssDNA fragments by heat. • Radioactive (32P) label to 5' end of DNA fragments with kinase reaction. • Cleave DNA strand at random nucleotides using snake venom. • DNA fragments applied to cellulose acetate strip and differently sized DNA strand fragments separated by size in electric field. • Fragments ending with G & T move to cathode • Fragments ending with C & A move to anode • Longest spots are connected to next longest and so on, indicating increasing number of nucleotides.

  13. Wandering Spot Analysis PROS: Used DNA rather than RNA. Higher accuracy. CONS: Poor results. Lots of work. Difficulty scaling to genomes. Requires X-rays and radiolabeling.

  14. Chemical Cleavage Method • Maxam and Gilbert (1977) • Similar to Wandering Spot Analysis. • Allowed for “at least 100 bases”. • lac Operator • 5’--G G C A C G A C A G G T T T C C C G A C T G G A A A G C G G G C A G T G A G C G C A A C G C A A T T A A T G T G A G T T A G G A C C G T G C T G T C C A A A G G G C T G A C C T T T C G C C C G T C A C T C G C G T T G C G T T A A T T A C A C T C A A--3'

  15. Chemical Cleavage Method • dsDNA fragments denatured into ssDNA fragments by heat. • Radioactive (32P) label to 5' end of DNA fragments with kinase reaction. • Cleave DNA strand at specific nucleotides using specific chemicals. (G>A, A>G, C, and C+T in four reaction tubes). • Differently sized DNA strand fragments separated by size via electrophoresis. • Fragments are found by means of autoradiographic detection of locations of radioactivity in form of dark spot. • Fragments ordered by size determine the sequence of the DNA molecule.

  16. Chemical Cleavage Method PROS: Was more popular than Sanger sequencing. Purified DNA could be used directly. (dsDNA) CONS: Lots of work. Difficulty scaling to genomes. Requires X-rays and radiolabeling.

  17. Chain Termination Method • Sanger et al. (1977) • Precursor to modern Sanger Sequencing (cycle sequencing) • Bacteriophage ϕX174 • First complete DNA genome sequenced

  18. Chain Termination Method • The dsDNA fragment is denatured into ssDNA fragments by heat. • ssDNA is multiplied into millions of copies. • Primer that corresponds to each fragment is attached. • Fragments are added to four polymerase solutions, each solution containing the four types of dNTPs but only one type of ddNTP. • Chain elongated until a termination nucleotide is randomly added. • Resulting dsDNA fragments are denatured to obtain a series of ssDNA of various lengths. • Fragments are separated by electrophoresis and termination dyed sequence is read.

  19. Chain Termination Method PROS: PCR errors overcome. Long sequences (~450 bp) CONS Only 1 sequence at a time Requires lots of DNA Expensive 2¢/base

  20. Fun Fact • 1973 cost/bp = $10,000+ • 2019 cost/bp = $0.000000014 • 1973 cost/genome = $64,000,000,000,000+ • 2019 cost/genome = $1,301

  21. References • https://www.britannica.com/science/DNA-sequencing • https://binf.snipcademy.com/lessons/dna-sequencing-techniques/maxam-gilbert • https://www.nature.com/scitable/topicpage/dna-sequencing-technologies-690/ • http://symposium.cshlp.org/content/18/123 • https://www.nature.com/articles/nrg2934 • https://www.pnas.org/content/70/12/3581 • https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=3630 • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC427284/pdf/pnas00139-0311.pdf • http://cdn.intechopen.com/pdfs/35675/intech-dna_representation.pdf • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC392330/pdf/pnas00024-0174.pdf • https://science.sciencemag.org/content/291/5507/1195 • https://www.promegaconnections.com/tag/chain-termination-method/ • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC431765/pdf/pnas00043-0271.pdf • http://www.cs.cmu.edu/~sssykim/teaching/f13/slides/NextGenSeq.pdf • http://www.aun.edu.eg/molecular_biology/workshop%20Realtime%202017/3-%20sequ.%20inter%20final.pdf • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC431765/ • https://www.genome.gov/about-genomics/fact-sheets/DNA-Sequencing-Costs-Data • https://www.veritasgenetics.com/our-thinking/whole-story • https://www.researchgate.net/figure/The-Sanger-sequencing-method-in-7-steps-1-The-dsDNA-fragment-is-denatured-into-two_fig2_234248746 • https://www.genscript.com/molecular-biology-glossary/1381/homochromatography • https://www.genetics.org/content/162/2/527.figures-only • https://www.ncbi.nlm.nih.gov/pubmed/14933251 • https://www.ncbi.nlm.nih.gov/pubmed/13580867?dopt=Abstract • https://academictree.org/chemistry/publications.php?pid=4333 • http://www.bioscirep.org/content/24/4-5/237 • https://www.sigmaaldrich.com/technical-documents/articles/biology/sanger-sequencing.html • http://biomodel.uah.es/metab/expresion/operon_Lac.en.htm • https://www.semanticscholar.org/paper/Advanced-Methods-in-Protein-Microsequence-Analysis-Wittmann-Liebold-Salnikow/5b52f7ff3e13a575bce3f83d1cba2f5100d9eb34/figure/11 • http://www.chemistryexplained.com/Ru-Sp/Sanger-Frederick.html

  22. Questions? sbeuchelt@unm.edu

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