Sistematika Molekular

Biologi Molekular
Evolusi molekular  Sistematika
Genetika Populasi
 Molecular systematics: Klasifikasi Molekular

 Modern classification is based on Genotypic

information
 Potential of unravelling the phylogeny of life (E.
Zuckerkandl & L. Pauling, 1965).
Based on information contents, biomolecules fall into 3
categories:
a. Semantides: (Primary semantides: DNA; Secondary semantides: RNA;
Tertiary semantides: Protein).
b. Episemantide: synthesized under the control of tertiary semantides:
ATP, carotenids (Chemotaxonomic markers)
c. Asemantides: not produced by the organisms and do not express any
of the information of the organisms, such as: exogenously supplied
vitamins, phosphate ions and oxygen.
 Karakterisasi Genomik:
Analisis Asam nukleat

1. DNA/RNA Fingerprinting (Sidikjari DNA)

2. Nucleic acid Hybridization
3. Phylogeny Tree: DNA sequencing
1. DNA/RNA Fingerprinting

1.1.BRENDA (Bacterial Restriction Endonuclease

Nucleic acid Digest Analysis)
1.2.RFLP (Restriction Fragment Length Polymorphism)
1.3.Ribotyping
1.4.Low molecular-weight-RNA profiles
1.5.ARDRA (Amplified Ribosomal DNA Restriction
Analysis)
1.6.RAPD (Random Amplified Polymorphic DNA)
1.7.Oligonucleotide Probes
1.8.Plasmid profiles and fingerprinting
Elektroforesis
DNA Fingerprinting
DNA Fingerprinting
METODE RFLP
METODE RAPD
RIBOTYPING
Ribotyping
Ribotype clustering anlysis
 2. Nucleic acid Hybridization
2.1. 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% (ΔTm < 5oC)
• 5’CCGTACCGTA3’

• 3’CCGTACCTGC5’
DNA Hybridization
3. Phylogeny Tree : DNA sequences

3.1. Teori evolusi (evolusi molekular)

3.2. Phylogenetic marker molecules
(molecular chronometer)
3.3. Amplifikasi rDNA: Teknik PCR
3.4. DNA sequencing
3.5. Sequence alignment
3.6. Konstruksi Phylogeny tree
3.7. Algoritme evolusioner
Phylogenetic Relation
Phylogeny Tree of life

Rooted Tree Unrooted Tree

3.1.Teori Evolusi
Evolusi: perubahan frekuensi gen/genotip
dalam suatu populasi
(Hukum Hardy-Weinberg)
Faktor evolusi:
• Mutasi
• Seleksi alam
• Aliran gena
• Genetic drift
Evolusi molekular
Genetika populasi
 3.2. 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 (Japan), EMBL
(Europe), Gene bank (USA)
Establish relationship among organisms
Phylogenetic marker :16S rRNA
Molekul 16S rDNA
Analisis 16S rDNA
Strategi Metode analisis
Isolasi & purifikasi DNA kromosomal

Amplifikasi 16S rDNA (gen 16S rRNA)

Sequencing 16S rDNA

Sequence alignment

Phylogeny tree construction
 3.3. 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
Siklus amplifikasi ruas DNA dengan PCR
Siklus PCR
Diagram PCR
RT-PCR
Produk PCR: 16S rDNA

1.5 Kb

Elektroforesis
3.4. 16S rDNA 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 !!!
Sintesis DNA
Molekul ddNTP
Metode dideoksi
Metode Dideoksi
Contoh DNA sequence
DNA sequence
a. CCAAGGCCTTAATTGGCC
b. CCATGGCCTTACTTGGCCGGCCTT
 d. Sequence alignment

1. GGGCUACACACGUGCUACAAUGGCGCA
2. GGGCUACACACGUGCUACAAUGGCGCA
3. GGGCUACACACGUGCUACAAUGGCGUA
4. GGGCUACACACGUGCUACAAUGGCGCA
5. GGGCGACACACGCGCUACAAUGGGCGG
****4** **** *4******** **4434
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
Algoritme evolusioner: clustering-Phylip (Phylogenetic
Inference Package)
1. Diskrit:
• Neigbour-joining (Saitou & Nei, 1987)
• Least square (Fitch & Margoliash, 1967)

2. Non-diskrit:
• Maximum Likelihood (Kluge & Farris, 1969)
• Maximum Parsimony (Felsenstein, 1981)

3. Nucleotide similarity & difference matrix (Chun, 2000) 

Phydit (Phylogenetic Editor)
Program Komputer:

PFE  preparasi data sequence

ClustalX  sequences alignment
PHYLIP (Phylogeny Inference Package):
• Phylogeny tree construction:
• Contain 4 evolutionary algorithm:
Neighbour Joining
Least square
Maximum Parsimony
Maximum Likelihood
Tree View
Words
 rRNA sequences of 4 strains
• Strain a: GCGGACAAA
• Strain b: GACGCCAAG
• Strain c: GAAAUCUAG
• Strain d: GAAAGCUAG
Hitung nilai distance antar strain !
“The distance : a single value based on the fraction of positions in which
the two sequences differ”
e.g. d(ab) = 4 ; d(ac) = ? ; d(ad) = ? ; d(bc) = ?
d(bd) = ? ; d(cd) = ?
 This table shows the sequence of a nine-base
region of the 16S rRNA of the four strains
Strain Site Number on DNA sequences
1 2 3 4 5 6 7 8 9
a G C G G A C A A A
b G A C G C C A A G
c G A A A U C U A G
d G A A A A C U A G
 Urutan kerja
• 1. Construct a distance matrix (First matrix) showing the
relatedness (as distance, d) between strains to determine
the most closely related strains, in this case between c
and d
• 2. Diagram the relatedness between the strains
• 3. Construct a second matrix to assess the distance
between the first paired group (cd) and the remaining
strains (a and b).
• 4. Since a and b are close to one another, determine and
diagram their relatedness.
• 5. Determine the relatedness between cd and a and b,
and diagram this.
First matrix
c
a b c
d
1
b dab = 4 - -
First tree

d cd/2 = 2/2
c dac= 6 dbc= 5 - = 1 unit

d dad= 5 dbd= 4 dcd=

2
Second Matrix
c
(cd) a
d
1
a
a d(cd)a = 11/2 -

b
2
b d(cd)b= 9/2 dab= 4
Second Tree

d(ab)/2 = 4/2
= 2 unit
 Final Tree
(11/2) +(9/2)
d(cd)(ab) = ----------------- = 2.5 unit
4

1
d

2
b
2.5
 FJ544332
PPK 6
FJ573187
FJ544356
AF290547
FJ527559
DQ207729
D16281
NR 024697
AB021192
AJ419629
NR 026138
NR 026144
X60615
EF688466
FJ823003
FJ715741
PSA 10
FJ544328
EU075147
NBRC12108
AB271751
NR 024691
AY043084
X60635
NR 024695
D16268
FJ573186
FJ544355
FJ544386
AJ276351
FJ544342
EU982547
PPK 5
NC 000964
X60605
NR 024689
CP000002
AY876289
NR 024690
AB271752
NR 026143
AJ316316
NR 026140
NR 026145
NR 026139
AJ302709
NR 026142
NR 026141
AB073190
0.1
Phylogeny tree

B. subtilis DSM 225

B. cereus DSM 226

L. casei DSM 227

C. perfringens DSM 228

C. tetani DSM 229

E. coli DSM 221