ANALISIS DNA:

PCR DAN
PENGGUNAANNYA
Rapid development of molecular biology was based on
three principles techniques :

Polymerase Chain Reaction (PCR)

DNA Sequencing

Cloning
 DNA EXTRACTION
DNA
Cell structure:
Endoplasmic reticulum

Golgi apparatus

nuclear DNA

Ribosome
mitochondrial
DNA (mtDNA)
Mitochondria
 Telomere

1p32.2
Short arm
(p)

Centromere

Long arm
(q)

Telomere
Human
Chromosome:
- 22 pair
autosomal
- 1 pair sex
Active (euchromatin) vs inactive (heterochromatin)
Nucleosome, basic building block of chromatin
 Principles of DNA Extraction

1. Preparasi sel/jaringan
Darah : digunakan leukositnya, eritrosit dilisiskan dan
dibuang. Secepatnya diekstraksi untuk mendapat
hasil optimal, penyimpanan sebaiknya pada suhu 4
derajat C, penyimpanan yang lama (> 1 bulan) akan
menurunkan hasil ekstraksi, volume 3 – 5 ml whole
blood
Jaringan : secepatnya diekstraksi. Penyimpanan
pada suhu -80 derajat. Jaringan yang disimpan
dalam paraffin blok sulit untuk diekstraksi
2. Lisis membran sel/organella (nukleus)
Phenol : senyawa yang sangat kuat untuk melisiskan
membran, tetapi toksik.

Guanidine isothicyanate : tidak toksik, digunakan

sebagai pengganti phenol

3. Denaturasi senyawa organik

Kloroform : paling banyak digunakan karena prosedur
sederhana, murah, mudah diperoleh

Proteinase K : denaturasi protein, perlu inkubasi

4. Presipitasi DNA
Isopropanol dengan volume 1:1

Ethanol absolut dan sodium asetat 1:10

5. Pencucian/washing
Ethanol 70%
 Methods of DNA Extraction

1. Phenol:chloroform
Phenol-choloroform-isoamyl alcohol
Metode standard untuk ekstraksi DNA

Akhir-akhir ini ditinggalkan, karena sifat toksik phenol

2. Salting Out
Menggunakan garam konsentrasi tinggi (NaCl 6 M)
Proteinase K untuk denaturasi protein
3. Guanidine isothiocyanate
Metode ini lebih cepat dibanding dua metode
sebelumnya
Thiocyanate bersifat toksik, untuk lisis dinding sel
Memerlukan chloroform untuk denaturasi protein

4. Silica Gel
Silica gel dapat mengikat DNA dengan perantaraan
garam/buffer tertentu (NaI)

Cepat, tetapi recovery DNA kurang

 Measuring DNA Quality

Kualitas DNA
Konsentrasi tinggi
Utuh, tidak terputus-putus
Tidak banyak terkontaminasi oleh protein

Menentukan Kualitas DNA

Spektrofotometer pada panjang gelombang 260 nm
(protein 280 nm; DNA baik apabila pengukuran pada
260:280 = 1,7 – 1,9). Satu OD = 50 ng DNA.
Dilihat dengan elektroforesis, band yang tebal secara
kualitatif menunjukkan DNA yang bagus
 Samples for DNA Extraction

1. Whole blood (leukosit : buffy coat)

Diambil leukositnya, sebelum itu eritrosit dilisiskan
dengan lisis buffer (EBL = erythrocyte lysis buffer).

Paling sering digunakan, 3 – 5 ml darah fresh cukup

banyak menghasilkan DNA

2. Jaringan biopsi/reseksi (otot, usus dll)

Sebelum ekstraksi, lebih dulu diinkubasi untuk
menghancurkan jaringan ikat
3. Amniotic fluid/Villi choriales
Untuk kepentingan prenatal diagnosis

Jumlah DNA yang diperoleh sangat sedikit

4. Jaringan lainnya
Untuk kepentingan forensik
Jaringan rusak/membusuk/terbakar

Folikel rambut, kuku, tulang dll

 Storing DNA

DNA disimpan dalam TE buffer (tris-hydroxymethyl

amino methana – EDTA)

Disimpan – 80 derajat bisa tahan bertahun-tahun

Untuk kerja, sebaiknya disiapkan DNA yang sudah

diencerkan menjadi 100 ng/mikroliter

Freezing – thawing berulang dapat meyebabkan

kerusakan DNA
POLYMERASE CHAIN REACTION (PCR)

DNA isolation/extraction usually produces a very small

amount of DNA concentration (several hundreds
nanogram / microliter)  difficult to analyze

It is necessary to amplify DNA / a fragment of DNA

PCR allows the production of more than 10 million

copies of a target DNA sequence from only a few
molecules
PCR REACTION MIXTURE

1. Template DNA
Usually the amount of template DNA is in the range of 0.01-1
ng for plasmid or phage DNA and 0.1-1 µg for genomic DNA,
for a total reaction mixture of 50 µl. Recently, with newest
PCR technology, as low as 1 ng of genomic DNA can be
amplified.
Higher amounts of template DNA usually increase the yield of
nonspecific PCR products
DNA Quality

All methods of DNA isolation (salting out, silica gel, phenol-

chloroform, guanidine isothiocyanate etc.) from fresh tissue
combine with good skills will provide high quality of DNA
 high concentration, pure, long DNA

Trace amounts of agents used in DNA purification

procedures (phenol, EDTA, Heparin, Proteinase K, etc.)
strongly inhibit Taq DNA Polymerase. Ethanol precipitation of
DNA and repetitive washing of DNA pellets with
70% ethanol is usually effective in removing traces of
contaminants from the DNA sample.
2. Primers
: oligonucleotide which is complement with the flanking
region of target DNA sequence
There are two primers; forward primer runs from 5’ to 3’ of
the sense template, reverse primer runs from 5’ to 3’ of the
antisense template
PCR primers are usually 15-30 (20 – 25) nucleotides in
length. Longer primers provide higher specificity.
The primer should not be self-complementary or
complementary to other primer in the reaction mixture, in
order to avoid primer-dimer and hairpin formation.
The melting temperature of flanking primers (forward and
reverse) should not differ by more than 5oC.
If the primer is shorter than 25 nucleotides, the approx.
melting temperature (Tm) is calculated using the following
formula: Tm= 4 (G + C) + 2 (A + T)
Annealing temperature should be approx. 5oC lower than
the melting temperature.
If the primer is longer than 25 nucleotides, the melting
temperature should be calculated using specialized
computer programs where the interactions of adjacent
bases, the influence of salt concentration, etc. are
evaluated.
The optimum annealing temperature should be
established from the experiments.
3. Deoxynucleoside triphosphates (dNTPs)  dATP,
dGTP, dCTP and dTTP)

The concentration of each dNTP in the reaction mixture is

usually 200 µM. It is very important to have equal
concentrations of each dNTP as inaccuracy in the
concentration of even a single dNTP dramatically increases
the misincorporation level.
4. Taq DNA polymerase

Heat stable DNA polymerase, isolated from hot spring

bacteria Thermus aquaticus found in Yellowstone National
Park, USA.
Usually 1-1.5 Units of Taq DNA Polymerase are used in
50 µl of reaction mix.
Higher Taq DNA Polymerase concentrations may cause
synthesis of nonspecific products.
If inhibitors are present in the reaction mix (e.g., if the
template DNA used is not highly purified), higher amounts
of Taq DNA Polymerase (2-3 U) may be necessary to
obtain a better yield of amplification products.
 5. MgCl2
The optimal concentration of MgCl2 has to be selected for
each experiment. Too few Mg2+ ions result in a low yield of
PCR product, and too many increase the yield of non-
specific products and promote misincorporation.
The recommended range of MgCl2 concentration is 1-4 mM
If the DNA samples contain EDTA or other chelators, the
MgCl2 concentration in the reaction mixture should be
raised proportionally.

Concentration of MgCl2
in 50 µl reaction mix, 1.0 1.25 1.5 1.75 2.0 2.5 3.0 4.0
mM

Volume of 25 mM
MgCl2, µl 2 2.5 3 3.5 4 5 6 8
6. PCR buffer
Standard PCR buffer contains 50 mM KCl, 10 mM Tris-
HCl, pH 8.3 at room temperature

PCR Mixture
All components should be added one by one in thin-wall
PCR tube carefully on ice  high probability of mistake
Many companies produced PCR mix which contains Taq,
MgCl2, dNTPs and PCR buffer in one reagents. Additional
components are template and primers only.
 Final Quantity, for 50 µl
Reagent
concentration of reaction mixture

Sterile deionized water - variable

10X Taq buffer 1X 5 µl

2 mM dNTP mix 0.2 mM of each 5 µl

Primer I 0.1-1 µM variable

Primer II 0.1-1 µM variable

Taq DNA Polymerase 1.25 u / 50 µl variable

25 mM MgCl2 1-4 mM variable*

Template DNA 10pg-1 µg variable

PCR Conditions

1. Initial Denaturation Step

The initial denaturation should be performed over an interval
of 1-3 min at 95oC. This interval should be extended up to
10 min for GC-rich templates.
The complete denaturation of the DNA template at the start
of the PCR reaction is of key importance. Incomplete
denaturation of DNA results in the inefficient utilization of
template in the first amplification cycle and in a poor yield
of PCR product.
2. Denaturation Step
Usually denaturation for 0.5-2 min at 94-95oC is sufficient,
since the PCR product synthesized in the first amplification
cycle is significantly shorter than the template DNA and is
completely denatured under these conditions.

3. Primer Annealing Step

Usually the optimal annealing temperature is 5oC lower
than the Tm; established based on experiments.
Incubation for 0.5-2 min is usually sufficient.
if nonspecific PCR products are obtained in addition to
the expected product, the annealing temperature should
be optimized by increasing it stepwise by 1-2oC.
4. Extending/Elongation Step
Usually the extending step is performed at 70-75oC. The
rate of DNA synthesis by Taq DNA Polymerase is highest at
this temperature.
Recommended extending time is 1 min for the synthesis of
PCR fragments up to 2 kb (or 1 kb). When larger DNA
fragments are amplified, the extending time is usually
increased by 1 min for each 1000 bp.

5. Final Extending Step

After the last cycle, the samples are usually incubated at
72oC for 5-15 min (7 min) to fill-in the protruding ends of
newly synthesized PCR products.
The terminal transferase activity of Taq DNA Polymerase
adds extra A nucleotides overhang to the 3'-ends of PCR
products.
Cycle Number

The number of PCR cycles depends on the amount of

template DNA in the reaction mix and on the expected yield
of the PCR product.
For less than 10 copies of template DNA, 40 cycles should
be performed. If the initial quantity of template DNA is
higher, 25-35 cycles are usually sufficient.
Denaturation

Annealing

Elongation
Gel electrophoresis of PCR product (amplicon)

Agarose gel is commonly used.

Reagents and equipment : gel frame (volume is 40 ml),
comb, agarose powder, ethidium bromide and buffer (TBE
= tris, boric acid, EDTA)
To make 2% agorose gel 40 ml, mix in erlenmeyer tube :
Agarose powder 0.8 gram
1 x TBE buffer 40 ml
Ethidium bromide 2 µl
Heat by microwave for 2 minutes, after cool down pour
the mixture on to gel frame equipped with comb.
Gel electrophoresis of PCR product (amplicon)

Mk 1 2 3

512 bp
Unspecific band