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MOLECULAR IDENTIFICATION OF MICROORGANISMS; PRINCIPLE & PROCESSESS OF 16S rRNA GENE SEQUENCING

ZUBAIRU UMAR DARMA DEPARTMENT OF MICROBIOLOGY FACULTY OF NATURAL AND APPLIED SCIENCES UMARU MUSA YAR’ADUA UNIVERSITY. MOLECULAR IDENTIFICATION OF MICROORGANISMS; PRINCIPLE & PROCESSESS OF 16S rRNA GENE SEQUENCING. January, 2018. Components of Bacteria cells. rRNA gene/rDNA (~1%).

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MOLECULAR IDENTIFICATION OF MICROORGANISMS; PRINCIPLE & PROCESSESS OF 16S rRNA GENE SEQUENCING

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  1. ZUBAIRU UMAR DARMA DEPARTMENT OF MICROBIOLOGYFACULTY OF NATURAL AND APPLIED SCIENCESUMARU MUSA YAR’ADUA UNIVERSITY MOLECULAR IDENTIFICATION OF MICROORGANISMS; PRINCIPLE & PROCESSESS OF 16S rRNA GENE SEQUENCING January, 2018

  2. Components of Bacteria cells rRNA gene/rDNA (~1%) phospholipids (~2%) Approx. 30% are cell components polysaccharides (~2%) Ions, small molecules (~4%) RNA (~6%) Approxi.70% are water proteins (~15%)

  3. Central Dogma Theory NB: rRNA Gene = rDNA

  4. REASONS rRNA GENES/rDNA ARE USED FOR BACTERIA IDENTIFICATION 1. All Bacteria have the genes 2. These genes perform similar function (protein synthesis) among all bacteria 3. High homology (are good for probing or PCR) 4. Tells us big picture lineages (has very large database sequence) 5. Rapidly detected by molecular biological method

  5. Bacteria Identification • Genomic DNA Extraction from Bacteria Cells • Gene Amplification • DNA Sequencing • Data BLAST & Neighbor Joining Tree Analysis

  6. DNA Extraction Cell lysis DNA extracts Bacteria Cells

  7. Cell lysis DNA RNA ions lipids proteins + + + + EDTA RNase A Proteinase K SDS EDTA

  8. Gene Amplification/PCR PCR Product (Amplified Target Gene) Target Gene DNA

  9. Structural components of the genes (Nucleotides) BASE BASE OCH2 P P P OCH2 P P P O O H H H H OH H OH OH deoxyribose NTP (dNTP) (Makes up DNA) ribose NTP (NTP) (Makes up RNA) BASE OCH2 P P P O H H H H dideoxyribose NTP (ddNTP) (Chain elongation inhibitor during Sequencing)

  10. A Existing DNA Strand O O G P O O C P O O T P Phosphodiester bond O H dNTP O T P P P O H O A P P P O H O C P P P O H

  11. DNA synthesis • Requirements: • DNA template • Primer: short oligonucleotide necessary for DNA polymerase to start • DNA polymerase: enzyme that constructs the DNA chain • deoxyribonucleotide triphosphates (dNTPs): building blocks of DNA A C C G G G A A G C C C C G G A T G A DNA polymerase A C G T DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  12. Steps involved in the synthesis • Step 1: Denaturation: separation of the two strands of the DNA duplex • Gyrase: Separates the two strands by pulling them apart • Helicase: breaks the hydrogen bonds that hold the two strands together gyrase helicase helicase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G gyrase

  13. Step 2: Annealing of primers to the DNA template strand • Primase synthesizes small complementary strands of RNA (“primers”) to the single strands of the DNA template primase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C G C C C C G primase G A T G A G C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  14. Step 3: Extension of newly constructed complementary DNA molecules • DNA polymerase adds bases to the ends of the primers, constructing an exact copy of the template DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  15. P P P P P P P O O O O O O O O O O O O O O P P P P P P P P P P P P P P A C G T G T C P P P P P P O O O O O O O O O O O O O T C G A T A A G C T A T G C A A G C T O O O O O O O O O O O O O O O O O O O O O O O O O O O O P P P P P P P P P P P P P P Summary of the DNA synthesis processes 1)DNA Template Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ 2)Primer 3)DNA Polymerase Mg2+ ions 4)dNTPs dNTPs 5)Mg2+ ions DNA Polymerase DNA Polymerase Primer Phosphodiester bond DNA Template

  16. T A G T A C T C A G T A Polymerase Chain Reaction • Polymerase Chain Reaction: cycling process consisting of the same 3 steps of DNA replication: • temperature cycling removes the need for other enzymes (gyrase/helicase, or primase) • PCR uses pre-made oligonucleotide DNA primers DNA polymerase gyrase primase helicase

  17. The Polymerase Chain Reaction • During PCR, a thermocycler brings the reaction mix to 3 different temperatures analagous to the 3 steps of DNA replication • Denaturation (95˚C) of the DNA template by heat • Annealing (45˚- 65˚C) of the primers to the template • Extension (72˚C) of the DNA strand by DNA polymerase • These steps are repeated for 30 cycles 95˚C 61˚C 72˚C denaturation annealing extension

  18. Thermocycler Program • Initial Denaturation: 95˚C 2 min • Start Cycle • Denaturation 91˚C 1 min • Annealing 45 - 65˚C 1 min • Extension 72˚C 1 min • Repeat Cycle 29 times (total = 30 cycles) • Final Extension 72˚C 5 min • Hold 4˚C ∞

  19. Denaturation • Denaturation occurs at 95˚C • The high temperature is used to break down the hydrogen bonds that hold the two strands together 95˚C G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  20. T A G T A C T C A G T A Annealing • Annealing occurs at 45˚- 65˚C • Oligonuclotide DNA primers anneal to their complementary sequences on the template strands • Annealing temperature depends on the melting temperature (Tm) of the primer (dependent on base composition) 95˚C 61˚C G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  21. Extension • Extension occurs at 72˚C • DNA polymerase attaches to the primers and extends the new DNA strand • The 3 steps (denaturation, annealing, and extension) are repeated for another 29 cycles 61˚C 72˚C DNA polymerase G A T G A G T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C DNA polymerase T C A G T A C A A C T G G C G T A A T C A T G G C C C T T C G G G G C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  22. Target Sequence • A desired target sequence is identified • To isolate the target sequence, primers that flank the region must be constructed • The DNA segment that is then amplified contains the region of interest Template DNA Forward Primer Reverse Primer Target Sequence of interest PCR Product

  23. PCR: Cycle 1 Denaturation Extension Annealing DNA Copies 4 Target Copies 0 Target Sequence of interest

  24. PCR: Cycle 2 Denaturation Extension Annealing DNA Copies 8 Target Copies 2

  25. PCR: Cycle 3 Denaturation Extension Annealing DNA Copies 16 Target Copies 8

  26. PCR: Cycle 4 Denaturation Extension Annealing DNA Copies 32 Target Copies 22

  27. PCR Amplification: within 35 cycles

  28. DNA Sequencing PCR product (Amplified Target Gene) Sequence of Target Gene DNA Sequencing AGCTGCTAAGCTTG AGCTTGCACAAGCT TAGCTTGCAAGCTT AGCTTGCAAGCTTG CAAGCTTGCAAGCT TGCAAGCTTGCAAG CTTGCAACGTTGCA AGCTTGCAAGCTTG AAGCTTGCAAGCTA

  29. DNA Sequencing • Dideoxy method of DNA sequencing (Sanger Method) • Single-stranded DNA to be sequenced serves as a template strand for DNA synthesis • single primer is used for DNA synthesis initiation • use of dNTPs along with labeled ddNTPs BASE BASE OCH2 OCH2 P P P P P P O O H H H H OH H H H dNTP ddNTP

  30. DNA Polymerization using ddNTPs A A O O O G P O G P O O O C P O C P O O O T P O C P O H O H O T P P P O T P P P O H H O A P P P O A P P P O H O H O C P P P Chain Termination O H

  31. Sequence Reaction • BigDye Terminator v3.1 Sequencing: • a Dye Terminator Cycle Sequencing Master Mix is used for sequencing reaction. Components include: • DNA polymerase I, Mg2+, buffer • dNTPs in ample quantities: • (dATP, dTTP, dCTP, dGTP) • ddNTPs in limited quantities, each labeled with a “tag” that fluoresces a different “color”: • (ddATP, ddTTP, ddCTP, ddGTP)

  32. Sequencing Reaction • Sequencing reaction is a cycled reaction using a thermocycler (as in the Polymerase Chain Reaction) • Like PCR, it consists of 3 steps: • Denaturation, Annealing, Extension; • these 3 steps are repeated for 30 cycles • Unlike PCR, it involves a single primer and labeled ddNTPs • extension proceeds normally until, by chance, DNA polymerase inserts a ddNTP, terminating the chain

  33. Sequencing Reaction DNA polymerase G A A C T C G G G T T T G G G T T T C C C C C C G G G T T T A A A C C C A A A A A A C C C T T T G G G G G G C C C G G G T T A A A A T T C A T G G C C C T T C G C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  34. G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T G T G T C C G T A C A A C T G G C G T G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T G T G T C C G T A C A A C T G G C G T A A T C A G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C G T G T C C G T A C A A C T G G C G T A A T C G T G T C C G T A C A A C T G G C G T A A T C A T G G G T G T C C G T A C A A C T G G C G T A G T G T C C G T A C A A C T G G C G T A A T C A T G G C G T G T C C G T A C A A C T G G C G T A A G T G T C C G T A C A A C T G G C G T A A T C A T G G C C G T G T C C G T A C A A C T G G C G T A A T C A T G T G T C C G T A C A A C T G G C G T A A T C A T G Sequencing Reaction G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G T G T C C G T A C A A C T G G C G G T G T C C G T A C A A C T G G C G T A A T DNA polymerase G A A C T C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  35. Sequencing Reaction G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C G T G T C C G T A C A A C T G G C G T A A T C A T G G C G T G T C C G T A C A A C T G G C G T A A T C A T G G G T G T C C G T A C A A C T G G C G T A A T C A T G G T G T C C G T A C A A C T G G C G T A A T C A T G T G T C C G T A C A A C T G G C G T A A T C A G T G T C C G T A C A A C T G G C G T A A T C G T G T C C G T A C A A C T G G C G T A A T G T G T C C G T A C A A C T G G C G T A A G T G T C C G T A C A A C T G G C G T A G T G T C C G T A C A A C T G G C G T G T G T C C G T A C A A C T G G C G DNA polymerase G A A C T C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  36. Electrophoresis • Electrophoresis is a technique used to separate DNA molecules on the basis of size and charge • Typical method used for analyzing, identifying and purifying DNA fragments

  37. Movement in an Electric Field • The mobility of molecules in the electrical field is also affected by their overall size or molecular weight Lower Molecular weight Higher Molecular weight – Agarose gel +

  38. DNA Gel Electrophoresis • These “markers” are run alongside samples, • Helps determine the length of the PCR sample • DNA fragments of the same length will migrate through the gel at the same rate 1500bp 1200bp 1100bp 1000bp 1050bp 900bp 800bp 820bp 700bp 650bp 600bp 500bp 400bp 400bp 300bp 280bp 200bp 100bp

  39. Electrophoresis • As the sample travels through the capillaries • Shorter fragments have less resistance and migrate faster • Longer fragments have more resistance and move slower - +

  40. Fluorescense detection • As fragments pass through detector window, the fluorescent “tag” of the ddNTP is excited by a laser • The emission of the “tag” is picked up by a detector and is translated to a colored peak unique to the nucleotide C T G A

  41. Sequencing Reaction G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G G T G T C C G T A C A A C T G G C G G T G T C C G T A C A A C T G G C G T A A T G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T G T G T C C G T A C A A C T G G C G T G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T T C G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C T G T G T C C G T A C A A C T G G C G T A A T C A G T G T C C G T A C A A C T G G C G T A A T C A T G G C C C G T G T C C G T A C A A C T G G C G T A A T C G T G T C C G T A C A A C T G G C G T A A T C A T G G G T G T C C G T A C A A C T G G C G T A G T G T C C G T A C A A C T G G C G T A A T C A T G G C G T G T C C G T A C A A C T G G C G T A A G T G T C C G T A C A A C T G G C G T A A T C A T G G C C G T G T C C G T A C A A C T G G C G T A A T C A T G T G T C C G T A C A A C T G G C G T A A T C A T G DNA polymerase G A A C T C C T A C T C A A G C A C A G G C A T G T T G A C C G C A T T A G T A C C G G G A A G C C C C G

  42. Capillary Electrophoresis • As the sample travels through the capillaries • Shorter fragments have less resistance and migrate faster • Longer fragments have more resistance and move slower - +

  43. Detection of fluorescent tags C G T A A A C A C G G C C C T T C G G G G C

  44. Final Data • Final data generated represents the complete chromatogram & the text version of the DNA sequence GCAGTCGAGCGGAACGAGTTATCTGAACCTTCGGGGAACGATAACGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATTGCCCTGATGTGGGGGATAACCATTGGAAACGATGGCTAATACCGCATAATAGCTTCGGCTCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAGGATATGCCCAGGTGGGATTAGCTAGTTGGTGAGGTAAGGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGA

  45. Sequence

  46. Sequenced data • GCAGTCGAGCGGAACGAGTTATCTGAACCTTCGGGGAACGATAACGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATTGCCCTGATGTGGGGGATAACCATTGGAAACGATGGCTAATACCGCATAATAGCTTCGGCTCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAGGATATGCCCAGGTGGGATTAGCTAGTTGGTGAGGTAAGGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCAGTGAGGAAGGTGGTGATGTTAATAGCATCATCATTTGACGTTAGCTGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCCG

  47. BLAST • BLAST: Basic Local Alignment Search Tool • www.ncbi.nlm.nih.gov/blast • Match the unknown sequences to the known published sequences in the GenBank web • Lists all sequences that produce significant alignments • Identification the genes of the organism’s genus or species and then • Identity the percentage of the alignment/match

  48. BLAST Results Summary: • C. sakazakii MM045 (More than 99% similarities) • Enterobacter sp. MM087 (100% similarities)

  49. References • Umar, Z.D., Azwady, N.A.A., Zulkifli, S.Z., and Muskhazli, M.(2016): Identification of Phenanthrene and Pyrene degrading Bacteria from used Engine Oil contaminated Soil. International Journal of Scientific and Engineering Research, 7(3): 680-686. • Salwa, H. T. 2012: Gene expression overview. • https://www.khanacademy.org/science/biology/gene-expression-central-dogma/central-dogma-transcription/a/nucleic-acids • https://www.khanacademy.org/science/biology/gene-expression-central-dogma/central-dogma-transcription/a/the-genetic-code-discovery-and-properties • Paul Billiet ODWS (2016). GENE EXPRESSION

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