molecular biology olimpiade biologi
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• DNA is negatively charged.
+-
Power
DNA
• When placed in an electrical field, DNA will migrate toward the positive pole (anode).
H
O2
• An agarose gel is used to slow the movement of DNA and separate by size.
Scanning Electron Micrograph of Agarose Gel (1×1 µm)
• Polymerized agarose is porous, allowing for the movement of DNA
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+-
Power
DNA
How fast will the DNA migrate?strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!*Small DNA move faster than large DNA…gel electrophoresis separates DNA according to size
smalllarge
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.PUTRA SANTOSO, BIO FMIPA
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wells Bromophenol Blue
Cathode(-)
Anode(+)
Gel
DNA(-)
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Ethidium Bromide requires an ultraviolet light source to visualize
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Visualizing the DNA (ethidium bromide)
100 200 300
1,650 1,000
500
850 650
400
5,000 bp 2,000
DNA ladder
DNA ladder
PCR Product
1 2 3 4 5 6 7 8
wells
+ - - + - + + -
Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA
Primer dimers
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100 200 300
1,650
1,000
500
850 650
400
12,000 bp
5,000
2,000
DNA Ladder Standard
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.
-
+
DNAmigration
bromophenol blue
Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule
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INTERPRETASI HASIL ELEKTROFORESIS
POLA PITA (BAND) MPK CERMINAN BERAT MOLEKUL (JARAK MIGRASI)
BM SAMA : JARAK MIGRASI SAMA (V. versa)
BM RINGAN : MIGRASI JAUH (V.versa)
JUMLAH PITA MENCERMINKAN JUMLAH SUBUNIT MOLEKUL YANG DIANALISIS
(EX.JML FRAGMEN DNA)
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1. Material DNA dari masing-masing individu dipotong dengan enzim restriksi. Hasil pemotongannya dipisahkan menggunakan agar elektroforesis sbb :
Maka pernyataan yang benar berkenaan dengan interpretasi hasil
Elektroforesis disamping adalah :
A. Kesemua individu adalah satu orang
B. Individu 3 mungkin adalah anak dari indv. 2 dan 4
C. Indv. 2 mungkin adalah anak dari 1 dan 3
D. Indv. 1 mungkin adalah anak dari 2 dan 3
E. Indv. 1 mungkin adalah anak dari 3 dan 4
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600
800
600
400
400
RX1 (BAM HI+HIindIII) : 800 bp dan 600 bp
RX2 (BAM HI+EcoRI) : 1200 bp dan 400 bp
RX3 (HIindIII+EcoRI) : 1400 bp dan1000bp, 400 bp PUTRA SANTOSO, BIO
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Teknik Blotting
Mendeteksi keberadaan fragmen-fragmen tertentu dari suatu material (DNA, RNA, protein, modified protein) dengan menggunakan detektor berupa probe yang komplementer dengan fragmen yang diinginkan
• Dasar teknik : elektroforesis dan fluorosensi
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BLOTTING TECHNIQUE
Southern blot : DNA
Northern blot : RNA
Western blot : Protein
Eastern blot : PTMP
Probe
DNA/RNA
DNA
atb
atbPUTRA SANTOSO, BIO FMIPA UNAND
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sisi restriksi ?1,2,3,4,5
sisi pengenalan probe 28s sisi pengenalan probe 18s
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Hap. A 1-2 : 1.5 kb
2-3 : 2.0 kb
3-4 : 1.0 kb
4-5 : 4 .0 kb
4.0
2.0
1.0
???
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BM Hap.A Hap.B Hap.C hap.D
8 kb
7 kb
6 kb
5 kb
4 kb
3 kb
2 kb
1 kb
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SEBAGIAN DNA TELAH HILANG PADA SEL TUMOR DIBANDINGKAN SEL NORMAL
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HIPOTESIS : PERUBAHAN PERKEMBANGAN PADA DUA JARINGAN DIATUR PD TKT TRANSKRIPSI
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SEL-SEL DITRANSFEKSI DENGAN PLASMID UTK SUATU PROTEIN REKOMBINAN (mRNA GEN RK---PROTEIN TOTAL--PROTEIN KHSUS)
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TERJADI PERUBAHAN PASCA TRANSLASI DARI PROTEIN (ANALISIS APA??)
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P1 : ACTIVATED BY STEROID
P2 : NO STEROID
mRNA panjang
mRNA pendek
BM .... migrasi??
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Smac : Second mitocondrial activating caspase
IAPs: Inhibitor Activating proteins
Apaf-1 : Apoptosis protein activating factor-1
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OPERON : GEN STRUKTURAL (SISTRON) + SITUS PENGONTROL + TERMINATOR
SEKUENSKONSENSUS
TERM
INAT
OR
OPERON POLISISTRONIK: OPERON DGN > 1 SISTRON
KUNCI : SEKUENS KONSENSUS (TITIK AWAL TRANSKRIPSI SETELAH SEKUENS TSB)PUTRA SANTOSO, BIO FMIPA UNAND
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Tumor suppressor gene atau anti-oncogene : gen yang melindungi sel terhadap perubahan ke arah kanker
Onkogen : gen yang jika terpicu akan menyebabkan sel berkembang menjadi kanker
Ekspresi gen supresor tumor bersifat resesif sedangkan onkogen bersifat dominan
Two-hit hypothesis oleh A.G Knudson
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GEN REPRESOR
GEN SUPRESOR
ONCOGEN
TUMOR DAN KANKER
TUMOR
KANKER
TWO HITS HYPOTHESIS
(SUPRESOR GENE)VS ONCOGEN
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Variable Number Tandem Repeat (or VNTR)
Schematic of a Variable Number of Tandem Repeats in 4 alleles.
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ALEL VNTR 1 VNTR 21 0.2 0.12 0.4 0.13 0.3 0.24 0.1 0.6
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Organisme A G C T
Escherichia coli 24,7 26,0 25,7 23,6
Khamir 31,3 18,7 17,1 32,9
Gandum 27,3 22,7 22,8 27,1
Salmon 29,7 20,8 20,4 29,1
Ayam 28,8 20,5 21,5 29,3PUTRA SANTOSO, BIO FMIPA UNAND
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NORTHERN BLOT WESTERN BLOT
TREATMENT : UPR
CONTROL : FPR
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