ACCURACY AND ACCURACY AND PRECISIONPRECISION

Sukarti MoeljopawiroSukarti MoeljopawiroLaboratorium BiokimiaLaboratorium BiokimiaFakultas BiologiFakultas Biologi

Accuracy and Precision

● Accuracy: May be defined as the degree of conformity to the truth

● Precision:● The degree of agreement between replicate

experiments

● Precision does not mean accuracy, since measurements may be highly precise but inaccurate due to a faulty instrument

Type of errors

● Errors may be:● Random● Careless● Inaccurate instruments

● Random error which are individually unpredictable

● Errors can arise from careless experiments work:● Using apparatus wrongly● Do not read the manufacture’s instructions● Using broken instruments

Standard and blank

● To obtain a value as accurate as possible from an estimation errors must be reduced to a minimum

● This can be done by:● Careful working● Using standard solution● Using a blank

● Standard solution:● Should be included in all measurements● Should be treated in an identical manner to the fluids

under investigation● Function: provides a useful check on the accuracy of a

method

Standard and blank (cont’d)

● Blank solution:● Should be included in any measurement● The same volume of distilled water replaces the

substance to be estimated● Should be treated in exactly the same way as the test

and standard● Function: provides a useful check on the reagents

purity

● The function of standard and blank: to correct the obtained value

Several blanks and controls need to be used when working with enzymes

Glassware

Macam Glassware● Pipet gondok (Volume pipette)● Pipet ukur (Graduated pipette)● Pipet tetes (Pasteur pipette)● Pipet mikro

● Labu godog (Digestion flask)● Labu ukur (Volumetric flask) ● Labu pemisah

● Gelas piala (Beaker glass) ● Botol timbang● Gelas arloji● Gelas ukur (Measuring cylinder)● Erlenmeyer (Erlenmeyer flask)

● Buret makro● Buret mikro● Buret semimikro

Cleaning glassware

● Grease rag soaked in chloroform or benzene and soaking overnight in chromic acid

● Very dirty apparatus soaking in a mixture of concentrated nitric and sulphuric acids

● After rinsing in tap water followed by several rinses in distilled water

● Normal glassware dried in an oven● Volumetric glassware rinsed with

alcohol then dried warm air

Terima kasih Terima kasih ……Terima kasih Terima kasih ……

CHROMATOGRAPHY

Sukarti MoeljopawiroLaboratorium BiokimiaFakultas Biologi

Separation of Compounds ofSeparation of Compounds ofBiological OriginBiological Origin

Using:● Extremes of temperature● Extremes of pH● Organic solvents● Oxidizing agents● Reducing agentshave to be avoided

Extreme physical conditions may:● Irreversibly change the structure of the

molecules● Destroy any biological activity

ChromatographyChromatography

This technique utilizes differences in the basic physical properties:

● Mass

● Size

● Shape

● Charge

● Adsorption effect

Chromatography Chromatography (cont’d)(cont’d)

A three component system is involved:

● The mixture to be separated (liquid)

● A stationary phase (column or film)

Solid play an active part in the separation process

● The mobile phase

Liquid (in most case) provides a medium for the molecules to move through

Kinds of ChromatographyKinds of Chromatography

● Gel Filtration

● Adsorption Chromatography

● Ion Exchange Chromatography

● Partition Chromatography● Paper Chromatography

● Thin-layer Chromatography

● Gas Chromatography● Gas-liquid Chromatography

● Gas-solid Chromatography

Gel FiltrationGel Filtration

Gel Filtration Gel Filtration (cont’d)(cont’d)

Adsorption ChromatographyAdsorption Chromatography

A and C : Association-dissociation phenomenaB : Solvation of soluteD and E : Interaction with adsorbent

Solute

Adsorbent

Solute Solvent SolventA B C

D E

Adsorption (chem.) : The taking up of one substance at the surface of another

Absorption (chem.) : Penetration of a substance into the body of another

Adsorbent (chem.) : The substance, either solid or liquid, on whose surface adsorption of another

substance takes placeChamber Dictionary of Science and Technology. Chambers Edinburgh.

Adsorption ChromatographyAdsorption Chromatography(cont’d)(cont’d)

Solvent reservoir

Filter paper disc

Adsorbent

Glass wool

Collecting tube

ANION exchanger with exchangeable

counter ions

CATION exchanger with exchangeable

counter ions

Ion Exchange ChromatographyIon Exchange Chromatography

––

–

–

–+

++

+

+ –

+

––

––

–

+

+

+

+

+

Ion Exchange ChromatographyIon Exchange Chromatography (cont’d)(cont’d)

Partition ChromatographyPartition Chromatography

Two kinds of Partition Chromatography:● Paper Chromatography

● Thin-layer Chromatography (TLC)

Partition Chromatography for the compounds that are soluble in both water and organic solvents

Adsorption Chromatography for the compounds that are readily soluble in organic liquid but sparingly soluble in water

Ion Exchange Chromatography for ionizable water soluble compounds

Paper ChromatographyPaper Chromatography

Based on direction of solvent flow:● Ascending Chromatography● Descending Chromatography● Circular Chromatography

Rf = Distance of migration of X

Distance moved by the solvent

Ascending ChromatographyAscending Chromatography

Descending ChromatographyDescending Chromatography

Circular ChromatographyCircular Chromatography

Thin-layer ChromatographyThin-layer Chromatography

● This method is very rapid (many separations can be completed under an hour)

● The spots are very compact (so it is possible to detect compounds at low concentration)

● Compounds separation is much better than paper chromatography

● Separated compounds can be detected using corrosive sprays at high temperature

Thin-layer ChromatographyThin-layer Chromatography(cont’d)(cont’d)

Thin-layer ChromatographyThin-layer Chromatography(cont’d)(cont’d)

Two DimensionalTwo DimensionalThin-layer ChromatographyThin-layer Chromatography

Two DimensionalTwo DimensionalThin-layer Chromatography Thin-layer Chromatography (cont’d)(cont’d)

Gas ChromatographyGas Chromatography

Gas Chromatography Gas Chromatography (cont’d)(cont’d)

● This method was first described by James and Martein (1952), and has been developed very rapidly

● This is the best method for separation of biological compounds

● Advantages of GC:● Very good separation● Time (analysis is short)● Small sample is needed (picogram)● Good detection system● Quantitatively analyzed

PrinciplesPrinciples

(gas)

STATIONARY PHASESTATIONARY PHASE

Sampleout

Samplein

(solid or heavy liquid coated onto a solid or support system)

MOBILE PHASEMOBILE PHASE

Gas Chromatography Gas Chromatography (cont’d)(cont’d)

Gas chromatography (GC) is a preferred method, only applicable to volatile substances

In GC mobile phase is gas, stationary phase could be:

Solid Gas Solid Chomatography (GSC)

Liquid Gas Liquid Chomatography (GLC)

In GLC, solid support is coated a liquid

Gas Chromatography Gas Chromatography (cont’d)(cont’d)

Gas chromatography (GC) consists essentially of a gas supply, column and detector

Gas Chromatography Gas Chromatography (cont’d)(cont’d)

● Gas supply consist of:● Cylinder of high purity gas under high

pressure● Gas could be nitrogen, helium, etc.

● Pressure regulation device● Flow regulation device● Flow measuring device

● Column:● Glass/stainless steel● Containing solid support (GSC)● Solid support is coated a liquid (GLC)

Gas Chromatography Gas Chromatography (cont’d)(cont’d)

● Detector is some device which generates a change in electrical signal in response to the solute as it comes off the column

● Most detectors require electronic amplification of the signal (electrometer)

● Kinds of detectors:● Flame ionization detector (FID)

● Nitrogen phosphorus detector (NPD)

● Electron capture detector (ECD)

● Flame photometric detector (FPD)

Schematic Diagram ofSchematic Diagram ofGas ChromatographyGas Chromatography

InstrumentationInstrumentation

● Injection port sample introduction● Manual - Direct Injection

● Automated - Autosampler

Instrumentation Instrumentation (cont’d)(cont’d)

● Oven Temperature Control● Isothermal

● Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Time (min)

Tem

p (

deg

C)

Instrumentation Instrumentation (cont’d)(cont’d)

● Column

Packed

Capillary

Instrumentation Instrumentation (cont’d)(cont’d)

● Detector● Destructive

● Mass Spectral (CI/EI)

● Flame Ionization (FID)

● Nitrogen-Phosphorus (NPD)

● Flame Photometric (FPD)

● Electrolytic Conductivity (Hall/ELCD)

● Non-destructive● Thermal Conductivity (TCD)

● Electron Capture (ECD)

● Photo Ionization (PID)

Instrumentation Instrumentation (cont’d)(cont’d)

● Detector● Biological detector

● Gypsy moth to detect Gypsy moth’s hormone● Human at the end of column detecting separated

aromas

● Normal detectors have been mentioned

Gas Chromatography SpectraGas Chromatography Spectra

Application of GC and TLCApplication of GC and TLC

Application of GC and TLCApplication of GC and TLC(cont’d)(cont’d)

… … Terima kasih …Terima kasih …

Sukarti MoeljopawiroBiochemistry Laboratory

Faculty of Biology

Polymerase Chain Reaction(PCR)

Polymerase Chain Reaction

• Ditemukan oleh Kary Mullis (1984)• Memperbanyak potongan DNA

PCR

• PCR terdiri atas beberapa proses:

– Pemanasan DNA untuk mendenaturasi DNA (memisahkan DNA menjadi 2 rantai tunggal)

– Penurunan suhu untuk penempelan primer oligonukleotida pada rantai DNA

– Polimerisasi karena adanya primer dan deoksiribonukleosida trifosfat serta adanya enzim

– Kemudian proses diulang lagi sampai 25 – 30 kali

SIKLUS PCR

Siklus PCR

DIAGRAM SIKLUS PCR

• Dapat menganalisis DNA yang sangat sedikit

• Kemampuan amplifikasinya besar

• Mampu memperbanyak DNA yang tidak segar/terdegradasi

• Spesifik, sederhana dan dapat menganalisis banyak sampel dengan cepat

• Mampu mendeteksi kelainan

KEUNGGULAN PCR

• Optimasi perlu waktu, kalau tidak optimum menyebabkan kesalahan interpretasi

• Karena sangat sensitif menyebabkan problem kontaminasi sangat tinggi terjadi kesalahan

• Kebersihan tanpa adanya kontaminasi dituntut sangat tinggi pada saat bekerja

KELEMAHAN PCR

• BAHAN:– DNA– Primers– Enzim (Thermostable Polymerase)– Empat macam deoksiribonukleosida trifosfat– Ion Mg– Buffer dan akuades

• PROSES TERDIRI DARI:– Denaturasi– Annealing– Polimerisasi– Kembali denaturasi

TEKNIK PCR

• Agar potongan DNA berhasil didapatkan dengan baik perlu optimasi:– Konsentrasi: enzim, dNTPs, Mg2+, primer

– Suhu dan waktu: annealing, denaturasi, polimerisasi/pemanjangan

• Konsentrasi enzim– 1 – 2,5 unit/100 μl larutan reaksi namun

tergantung dari template target dan primer. Masing-masing sampel perlu dioptimasi

– Konsentrasi untuk optimasi dianjurkan antara 0,5 – 5 unit/100 μl

OPTIMASI PCR

• Konsentrasi dNTPs

– Stok dNTPs harus netral (pH 7), disimpan dengan konsentrasi 10 mM pada suhu – 20°C. Konsentrasi keempat macam deoksiribonukleosida trifosfat harus sama pada larutan dNTPs

– Spesifitas dan ketelitian naik dengan rendahnya konsentrasi, sebab dengan konsentrasi yang rendah memperkecil kemungkinan terjadinya salah penempelan primer (± 20 μM/100 μl larutan reaksi).

Namun konsentrasi tersebut juga tergantung DNA yang diamplifikasi

OPTIMASI PCR (lanjt.)

• Konsentrasi Mg2+

– Optimasi konsentrasi ion Mg sangat penting sebab konsentrasi ion Mg akan mempengaruhi: penempelan primer, suhu dissosiasi baik jalin DNA template maupun produk PCR, spesifitas produk, pembentukan primer-dimer, aktivitas enzim dan ketelitian

– Konsentrasi sebaiknya antara 0,5 – 2,5 mM

• Konsentrasi primer– Konsentrasi primer dianjurkan antara 0,1 – 0,5 μM.

Semakin tinggi konsentrasi primer menyebabkan terjadinya salah tempel (mispriming) dan terjadi penumpukan produk yang tidak diinginkan, juga kemungkinan terjadi primer-dimer

OPTIMASI PCR (lanjt.)

– Rumus untuk mengirakan konsentrasi primer:

0,067 Y / OD260 = X

(Ruiz et al., 1997)

Y : volume reaksi (μl)X : volume primer yang akan dipakai (μl)OD260 : optical density pada λ 260 nm

OPTIMASI PCR (lanjt.)

• Suhu annealing

– Suhu yang diperlukan untuk annealing primer tergantung pada: komposisi basa, panjang dan konsentrasi DNA

– Suhu annealing terbaik biasanya 5°C dibawah Tm. Biasanya suhu antara 50 – 72 °C menghasilkan produk yang bagus

– Suhu terlalu tinggi menyebabkan penempelan primer tidak betul dan kesalahan perpanjangan pada ujung 3’ primer, sehingga suhu annealing sangat kritis lebih-lebih pada tahap putaran permulaan

OPTIMASI PCR (lanjt.)

• Pemanjangan rantai (primer extension)– Waktu pemanjangan tergantung pada panjang dan

konsentrasi DNA yang diamplifikasi serta suhu– Suhu pemanjangan umumnya 72 °C

Suhu 72 °C selama 1 menit cukup untuk produk dengan panjang 2 kb

• Suhu dan waktu denaturasi– Penyebab utama gagalnya reaksi PCR adalah tidak

sempurnanya proses denaturasi– Biasanya: 95 °C selama 30 detik atau

97 °C selama 15 detik Namun, lebih tinggi mungkin lebih baik

terutama DNA yang banyak pasangan G–C (Innes et al., 1990)

OPTIMASI PCR (lanjt.)

– Jika denaturasi tidak sempurna, mungkin DNA kembali berikatan ataupun tidak mampu menerima primer yang akan menempel, sehingga sensitifitas turun produk turun. Ini terjadi jika waktu denaturasi terlalu pendek. Jika waktu terlalu lama enzim polimerase akan rusak

Beberapa protokol menganjurkan denaturasi mula-mula 94 °C selama 1 – 10 menit (Ruiz et al., 1997)

– (Innes et al., 1990) menyebutkan waktu paruh polimerase DNA kurang dari 2 jam untuk 92,5 °C, 40 menit untuk suhu 95 °C, dan 5 menit untuk suhu 97,5 °C

OPTIMASI PCR (lanjt.)

• Jumlah putaran– Jumlah tergantung pada konsentrasi DNA mula-

mula dengan catatan semua parameter sudah dioptimasi

– Amplifikasi lebih dari 40 putaran akan menghasilkan produk PCR yang salah

– Terlalu sedikit putaran akan mendapatkan produk yang sedikit

– Rekomendasi putaran vs konsentrasi mula-mula:

Jumlah Molekul Awal Jumlah Putaran 3 x 105 25 sampai 30 1,5 x 104 30 sampai 35 1 x 103 35 sampai 40 50 40 sampai 50

OPTIMASI PCR (lanjt.)

VERIFIKASI HASIL PCR

• Mudah, cepat, jumlah sampel DNA yang dibutuhkan sedikit

• Tidak memerlukan pengetahuan tentang sekuen DNA yang diteliti

• Tidak melibatkan radioaktif

• Analisis RAPD untuk identifikasi penanda genetik (linked dengan suatu sifat khususnya sifat yang sulit diamati secara morfologi/fenotip)

• Teknologi RAPD mempunyai prospek baik untuk mengidentifikasi penanda DNA terkait dengan jenis kelamin tanaman

APLIKASI PCR UNTUK RAPDKeunggulan RAPD

Karena RAPD berdasarkan reaksi PCR maka optimasi komponen dan kondisi PCR sangat mempengaruhi hasil

Sehingga metode RAPD menuntut kondisi optimal untuk mengembangkan protokol yang reproducible

KELEMAHAN METODE RAPD

• Pemetaan genetik• Analisis filogenetik• Penyusunan peta keterkaitan (linked)• Evaluasi aliran gen antar jenis• Identifikasi:

– Individu– Kultivar atau jenis dengan sidik jari

genom– Tetua dalam analisis silsilah

PENGGUNAAN METODE RAPD

Identifikasi penanda kelamin dengan metode RAPD untuk mendeteksi

perbedaan molekular tanaman salak jantan dan betina pada tingkat DNA

(Parjanto, Disertasi UGM 2006)

CONTOH PENGGUNAAN RAPD

CONTOH PENERAPAN RAPD

300 -

400 -

600 -

bp

Gambar 1. Pola pita RAPD tanaman salak (S. zalacca) jantan (J1-J5) dan betina (B1-B5) menggunakan primer OPA-11. Tanda panah menunjukkan fragmen DNA 360 bp hasil amplifikasi dengan OPA-11 (OPA-11360) tidak spesifik pada satu jenis kelamin. M = marker 100 bp.

CONTOH PENERAPAN RAPD (lanjt.)

Gambar 2. Penanda RAPD (400 bp) spesifik pada tanaman salak (S. zalacca) jantan hasil amplifikasi dengan primer OPP-08 (OPP-08400). B = tanaman betina, J = tanaman jantan, M = marker 100 bp.

- 400

- 1000

- 600

bp

(Parjanto, Disertasi UGM 2006)

Terima Kasih