Abdul Rahman Siregar

Laboratorium Mikrobiologi, Fakultas Biologi UGM

4 September 2017
 Karakterisasi Genomik:
Analisis Asam nukleat

1. DNA/RNA Fingerprinting (Sidikjari DNA)

2. Nucleic acid Hybridization
3. Phylogeny Tree: DNA sequencing
 Based on information contents, biomolecules
fall into 3 categories:

Primary semantides: DNA; Secondary

Semantides semantides: RNA; Tertiary semantides:
Protein).

synthesized under the control of

Episemantides tertiary semantides: ATP, carotenoids
(Chemotaxonomic markers)

not produced by the organisms and do not

express any of the information of the
Asemantides organisms, such as: exogenously supplied
vitamins, phosphate ions and oxygen.
 DNA/RNA Fingerprinting
BRENDA
(Bacterial
Restriction
Endonuclease Ribotyping
Nucleic acid RAPD
RFLP
Digest Analysis) (Random
(Restriction
Fragment Amplified
Length Polymorphic
ARDRA DNA)
Plasmid Polymorphism (Amplified
profiles and Ribosomal DNA
fingerprinting Restriction
Low
Analysis)
molecular- Oligonucleo
weight-RNA tide Probes
profiles
Ribotyping (Group 1 10)
Diagramatic Representative
(Group 1 10)
Clustering (Group 1- 10)
Identifikasi mikrobia dengan sidikjari DNA
Identifikasi mikrobia dengan 16S rDNA
 2. Nucleic acid Hybridization
DNA-DNA hybridization: DNA-DNA relatedness
genomic species concept
A species: a group of strains characterized by a high
degree of similarity in DNA sequence and phenotypic
properties
Kelemahan:
Teknik hibridisasi relatif sulit
Reprodusibilitas rendah
Keunggulan:
Obyektif dan dapat dilakukan pada semua strain
Mencerminkan similaritas genom secara total
 Metode hibridisasi DNA

DNA-DNA relatedness: 70% (Tm < 5oC)

5CCGTACCGTA3 5CCGTACCTAG3
3GGCATGGCAT5 3GGCATGGATC5
Elektroforesis
Elektroforesis DNA
 Southern Blotting

Pemindahan pola band DNA pada gel ke

membran nilon
DNA Hybridization
Temp and Hybridization
Animasi Hibridisasi DNA dengan Probe
DNA hyb method
DNA Hybridization
method: colony hyb
Identifikasi pemilik DNA
 3. Phylogeny Tree
Phylogeny Tree dan interpretasinya
Marker filogenetik
Persyaratan marker filogenetik
Molekul rRNA sebagai marker filogenetik
Struktur molekul rRNA (rDNA)
Alignment sequence rRNA (rDNA)
Algoritme konstruksi pohon filogeni
Validitas topologi pohon filogeni
 Phylogeny Tree: DNA sequences

a. Phylogenetic marker molecules (molecular

chronometer)
b. Amplifikasi rDNA: Teknik PCR
c. DNA sequencing
d. Sequence alignment
e. Konstruksi Phylogeny tree
f. Algoritme
 Teori Evolusi
Evolusi perubahan frekuensi gen/genotip
dalam suatu populasi (Hukum Hardy-Weinberg)
Faktor evolusi:
Mutasi
Seleksi alam
Aliran gen
Genetic drift
Evolusi molekular
Genetika populasi
 a. Phylogenetic marker molecules
(molecular chronometer)
Ubiquitously distributed and highly conserved
Functionally equivalent
Homologous housekeeping i.e. vital molecules
16S rDNA & 18S rDNA genes (Woese, 1978)
Sequences are deposited in database: DDBJ (DNA
Data Bank of Japan), EMBL (The European
Molecular Biology Laboratory), NCBI (National
Center for Biotechnology Information - USA), etc
Establish relationship among organisms
 b. Amplifikasi rDNA: Teknik PCR

PCR: Polymerase Chain Reaction

In vitro replication of DNA

Komponen reaksi PCR:

DNA template
Primer (Forward & Reverse)
dNTP (dATP, dGTP, dCTP, dTTP)
Taq polymerase
Mg++
Buffer
Inkubasi (Thermocycler): siklus PCR

Denaturasi (96oC) 1 menit

Annealing (54oC) 1 menit
Extension (72oC) 2 menit
 Phylogenetic Classification

1. Isolasi dan purifikasi DNA kromosomal

2. Amplifikasi gen 16S rDNA/18S rDNA (PCR)
3. DNA sequencing : metode dideoksi (Sanger-Coulson)
4. Preparasi data DNA sequences
5. DNA sequences alignment (Clustal-X)
6. Konstruksi Phylogeny Tree (Phylip)
7. Visulaisasi Phylogeny Tree (TreeView)
8. Editing & Presenting Phylogeny Tree (Words)
16S rRNA
Molekul 16S rDNA
Siklus amplifikasi ruas DNA dengan PCR
Siklus Amplifikasi DNA: PCR
DNA Amplification: PCR
Animasi PCR
PCR Product of 16S rDNA
Identifikasi Produk PCR
c. DNA sequencing: metode dideoksi
 c. DNA sequencing
Metode Dideoksi (Sanger & Coulson)
Campuran dNTP dan ddNTP:
a. dATP, dGTP, dCTP, dTTP dan ddATP
b. dATP, dGTP, dCTP, dTTP dan ddGTP
c. dATP, dGTP, dCTP, dTTP dan ddCTP
d. dATP, dGTP, dCTP, dTTP dan ddTTP

ddNTP: tidak ada OH pada atom C no. 3

sehingga tidak dapat menambahkan
nukleotida berhenti sintesis DNA !!!
DNA sequencing: metode dideoksi (Manual)
DNA sequencing: metode dideoksi (Manual)
DNA sequence chromatogram
DNA sequence
 d. Sequence alignment (Clustal-X)

1. GGGCUACACACGUGCUACAAUGGCGCA
2. GGGCUACACACGUGCUACAAUGGCGCA
3. GGGCUACACACGUGCUACAAUGGCGUA
4. GGGCUACACACGUGCUACAAUGGCGCA
5. GGGCGACACACGCGCUACAAUGGGCGG
* * * * 4 * * * ** * * 4 * * * * * * * * * * 4 4 3 4

To align: to ensure that only homologous nucleotides are arranged in

columns and consequently are recognized as being identical or
different.
 e. Konstruksi Phylogeny tree

1. Software: Program Phylip

(Phylogentic Inference Package)
Input: Data sequence DNA (rDNA) yang sudah dialligned
Output: Phylogeny Tree
sebagai dasar klasifikasi
sebagai dasar identifikasi
2. Software: Phydit (Phylogenetic Editor: Chun, 2000)
Input: alligned DNA sequences
Output: nucleotide similarity & difference matrix
sebagai dasar identifikasi
f. Algoritme Konstruksi Phylogeny Tree :
Phylip

1. Diskrit:
Neigbour-joining (Saitou & Nei, 1987)
Least square (Fitch & Margoliash, 1967)

2. Non-diskrit:
Maximum Likelihood (Kluge & Farris, 1969)
Maximum Parsimony (Felsenstein, 1981)
Phylogeny Tree
Tree of Life: Three Domain
Tree of Life: Three Domain
Bacterial phylogeny
 T
S. galbus DSM 40089 (X79852)
T
S. hygroscopicus subsp. limoneus ATCC 21431 (X79853)
T
S. cellostaticus DSM 40189
T
52 S. lincolnensis NRRL 2936 (X79854)
T
S. curacoi DSM 40107
* 97 T
S. chartreusis DSM 40085 (AJ 399468)
* S.violaceusniger NRRL-ISP T5182 (AJ 391815)
S. scabies ATCC 49173 (D63862)
T
S. bottropensis ATCC 25435 (D63868)
T
S. neyagawaensis ATCC 27449 (D63869)
T
S. diastatochromogenes ATCC 12309 (D63867)
T
S. luteoreticuli ISP 5509 (X53172)
T
S. acidiscabies ATCC 49003 (D63865)
T
S. ornatus DSM 40307 (X79326)
T
S. cyaneus DSM 40108 (AJ 399471)
T
S. griseus subsp. griseus ATCC 23345 (AF056711)
T
S. bikiniensis DSM40581 (X79851)
T
S. clavuligerus DSM 40751
T
S. lavendulae IFO 12789 (D85116)
T
S. lavendulae subsp. lavendulae DSM 2014 (X53173)
T
71 S. subrutilus DSM 40445 (X80825)
T
S. virginiae IFO 12827 (D85123)
* T
S. lavendulae subsp. fuscus IFO 14028 (D85114)
T
S. phosalacineus JCM 3340 (U93330)
T
S. brunneus IFO 14627 (U93314)
T
S. paracochleatus IFO 14769 (U93328)
T
S. setae JCM 3304 (U93332)
T
100 S. cystargineus JCM 7356 (U93318)
T
S. aureofaciens NRRL 2209 (Y15504)
* T
S. kifunensis JCM 9081 (U93322)
T
S. azaticus IFO 13803 (U93312)
T
S. purpureus DSM 43460 (X53170)
T
S. ladakanum DSM 40587 (X53167)
T
S. baldacci DSM 40845 (X53164)
T
S. olivoreticuli DSM 40105 (X53166)
T
S. cinnamoneus ISP 5005 (X53171)
T
S. abikoensis DSM 40831 (X53168)
T
S. salmonis DSM 40895 (X53169)
T
S. griseocarneus DSM 40004 (X99943)
T
S.. albus subsp. albus DSM 40313 (X53163)
T
S. violaceusniger NRRL-ISP 5563 (AJ 391823)
T
S. violaceusniger NRRL B-1476 (AJ 391822)
95 S. violaceusniger NRRL B-1356 (AJ 391812)
* S. hygroscopicus NRRL 2339 (AJ 391821)
T
S. hygroscopicus NRRL 2387 (AJ 391820)
S. hygroscopicus NRRL B-1477 (AJ 391819)
T
94 S. melanosporofaciens NRRL B-12234 (AJ 391837)
Streptomyces sp. strain.76
* S. violaceusniger NRRL B-1865 (AJ 391818)
T
S. malaysiensis DSM 41697 (AF117304)
S. hygroscopicus subsp. geldanus NRRL 3602 (AJ 391824)
T
S. chrestomyceticus ISP 5545
80 T
S. lydicus ATCC 25470 (Y15507)
* * S.violaceusniger NRRL 8097 (AJ 391816)
T
S. mashuensis DSM 40221 (X79323)
T
S. sparsogenes NRRL 2940 (AJ391817)
T
S. odorifer DSM 40347 (Z76682)
T
S. felleus DSM 40130 (Z76681)
T
100 S. canescens DSM 40001 (Z76684)
T
S. albidoflavus DSM 40455 (Z76676)
* T
62 S. sampsonii ATCC 25495 (D63871)
T
S. rutgersensis DSM 40077 (Z76688)
* T
S. gougerotii DSM 40324 (Z76687)
T
S. intermedius DSM 40372 (Z76686)
T
S. diastaticus DSM 40496 (X53161)
T
S. ambofaciens ATCC 23877 (M27245)
T
S. tendae ATCC 19812 (D63873)
T
S. eurythermus ATCC 14975 (D63870)
T
S. caelestis NRRL 2418 (X80824)
T
S. brasiliensis DSM 43159 (X53162)
57 S. violaceusniger NRRL 2834 (AJ 391813)
* . S. violaceusniger NRRL B-5799 (AJ 391814)
85 T
S. thermovulgaris DSM 40444 (Z68094)
T
* S. alkalitolerans TA 56 (AJ000284)
52 T
S. thermogriseus CCTCCAA 97014 (AF056714)
T
* S. thermoviolaceus DSM 41392 (Z68095)
T
S. thermoviolaceus subsp. violaceus DSM 40443 (Z68096)
T
S. thermodiastaticus DSM 40573 (Z68101)
T
S. bluensis ISP 5569 (X79324)
100 T
S. megasporus DSM 41476 (Z68100)
T
* S. macrosporus DSM 41449 (Z68099)

0.01

Figure 4.4. Neighbour-joining tree (Saitou & Nei, 1987) showing relationships between marker strains and representatives of the genus
Streptomyces based on nearly complete 16S rDNA sequences. The asterisks denote the branches that were also recovered using the
least-squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981) and maximum- parsimony (Kluge & Farris, 1969)
treeing algorithms. The numbers at the nodes indicate the levels of bootstrap support (%) based on a neighbour-joining analysis of 1000
resampled data sets. The arrow indicates the estimated root position of the tree. The scale bar indicates one nucleotide substitution per
100 nucleotides in 16S rRNA gene sequences.
 NT similarity table
A B C D

A 2/1451 5/1434 9/1356

B 99,98 18/1457 25/1467

C 99,95 99,78 28/1455

D 99,80 99,70 99,65

Phydit: Phylogenetic Editor