1 / 22

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.

emily
Download Presentation

First Sequencing Methods

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related