Molecular biology olimpiade biologi

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CELLS AND MOLECULAR

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PUTRA SANTOSO, BIO FMIPA UNAND

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buffer

Cathode(negative)

Anode(positive)

wells

DNA


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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

UNAND

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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


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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

FMIPA UNAND

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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

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atbPUTRA SANTOSO, BIO FMIPA UNAND

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ENZIM RESTRIKSI


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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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WESTERN ANALYSIS


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GEN RICIN DARI Ricinus communisPUTRA SANTOSO, BIO FMIPA

UNAND

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LIGASE


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APOPTOSIS (CDP)

NECROSIS


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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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CANCERTUMOR


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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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NEW TRAND IN CANCER THERAPHY


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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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POINT MUTATION


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MIKROBA TERMOFILIK


Molecular biology olimpiade biologi

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