Dasar-dasar Pemeriksaan Biologi Molekuler

Dasar-dasar Pemeriksaan Biologi
Molekuler
TRI SETYAWATI
3 teknik dasar dalam biologi molekuler
Polymerase Chain Reaction (PCR)
DNA Sequencing
Cloning
EkstraksiDNA
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
Jumlah kromosom
manusia:
- 22 pair
autosomal
- 1 pair sex
Eukromatin (aktif) dan heterokromatin (inaktif)
Nukleosom, dasar pembentukan kromatin
Prinsip dalam ekstraksi DNA
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%
Metode ekstraksi DNA
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
Pengukuran Kualitas DNA
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
Sampel untuk Ekstraksi DNA
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
Penyimpanan 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)
Adalah teknik penggandaan DNA/amplifikasi DNA
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, phenolchloroform, 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. Deoxynucleotide 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,
mM
Volume of 25 mM
MgCl2, µl
1.0
1.25
1.5
1.75
2.0
2.5
3.0
4.0
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
concentration
Quantity, for 50 µl
of reaction mixture
-
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
1.25 u / 50 µl
variable
25 mM MgCl2
1-4 mM
variable*
Template DNA
10pg-1 µg
variable
Reagent
Sterile deionized water
Taq DNA Polymerase
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, 2535 cycles are usually sufficient.
Denaturation
Annealing
Elongation
Animasi PCR
http://www.youtube.com/watch?v=v4L7rvmBXbY
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
RESTRICTION ENDONUCLEASE
A restriction enzyme (or restriction endonuclease) is an enzyme
that cuts double-stranded DNA. The enzyme makes two incisions,
one through each of the sugar-phosphate backbones (i.e., each
strand) of the double helix without damaging the nitrogenous
bases  cleave the sugar-phosphate backbone of DNA
Thousands of restriction enzymes have been isolated from
bacteria, where they appear to serve a host-defense role.
Restriction enzymes are classified biochemically into four types
(classes), designated Type I,Type II, Type III, and Type IV.
Type I and III, both the methylase and restriction
activities are carried out by a single large enzyme
complex. Both require ATP for their proper function.
In type II systems, the restriction enzyme is independent of
its methylase, and cleavage occurs at very specific sites that
are within or close to the recognition sequence. The vast
majority of known restriction enzymes are of type II, and it is
these that find the most use as laboratory tools. The first to
be discovered and utilized was EcoRI, which is staggered and
its recognition sequence is 5'-GAATTC-3'. Most type II
enzymes cut palindromic DNA sequences
In type IV, the restriction enzymes target only
methylated DNA.
Restriction enzymes are named based on the bacteria in which
they are isolated in the following manner:
E : Escherichia (genus); co : coli (species); R : RY13 (strain); I : First
identified Order ID'd in bacterium
The substrates for restriction enzymes are specific sequences of
double-stranded DNA called recognition sequences.
The length of restriction recognition sites varies: The enzymes
EcoRI, SacI and SstI each recognize a 6 base-pair (bp) sequence
of DNA, whereas NotI recognizes a sequence 8 bp in length,
and the recognition site for Sau3AI is only 4 bp in length.
Different restriction enzymes which have the same recognition
site are called isoschizomers (SacI and SstI)
Restriction recognitions sites can be unambiguous or
ambiguous: BamHI recognizes the sequence GGATCC
unambiguous. HinfI recognizes a 5 bp sequence starting with
GA, ending in TC, and having any base between (in the table, "N"
stands for any nucleotide)  ambiguous recognition site. XhoII
also ambiguous)
The recognition site for one enzyme may contain the restriction
site for another. The BamHI recognition site contains the
recognition site for Sau3AI.
most recognition sequences are palindromes - they read the
same forward (5' to 3' on the top strand) and backward (5' to
3' on the bottom strand).
Pattern of DNA Cutting by Restriction Endonuclease
1. 5' overhangs: The enzyme cuts asymmetrically within the
recognition site such that a short single-stranded segment
extends from the 5' ends. BamHI cuts in this manner.
The 5' or 3' overhangs generated by enzymes that cut
asymmetrically are called sticky ends or cohesive ends, because
they will readily stick or anneal with their partner by base
pairing.
2. 3' overhangs: asymmetrical cutting within the recognition site,
the result is a single-stranded overhang from the two 3' ends. KpnI
cuts in this manner.
3. Blunts: Enzymes that cut at precisely opposite sites in the two
strands of DNA generate blunt ends without overhangs. SmaI is an
example of an enzyme that generates blunt ends.
PCR – RFLP (Restriction Fragment Length Polymorphisms)
A combination of PCR – restriction method to detect SNP
(single nucleotide polymorphsim)
The sample is first run in a restriction digest to cut the DNA, then
gel electrophoresis is performed on this digest. In the case of
MTHFR C677T polymorphism, single band of 198 bp denotes CC
genotype, two bands of 198 and 175 bp denote CT genotype
and single band of 175 bp denotes TT genotype.
After gel electrophoresis, DNA can be visualized by staining
with ethidium bromide, an intercalating agent and fluorescent
dye.
PCR amplification of MTHFR exon 4
G A N T C
C T N A G
198 bp
G A G C C
Ala
Enzyme digestion (HinfI)
CC (wild type)
TT (mutant)
~23 bp
G A G T C
Val
~175 bp
Gel Electrophoresis
M
CC
CT
TT
198 bp
175 bp
PCR-RFLP untuk polimorfisme G135A gena RET
PCR amplification of RET exon 2
Enzyme digestion (EagI)
C G G C C G
G C C G G C
294 bp
~87 bp
~207 bp
Gel Electrophoresis
Vietnamese SMA Patients
Mk
A
3
5
7
8
9
11 19 20 21
22 23
24 C+ CSMN1 Exon 7
SMN2 Exon 7
B
C
SMN2 Exon 8
SMN2 Exon 8
SMN2 Exon 8
NAIP Exon 5
DNA SEQUENCING
The term DNA sequencing encompasses biochemical methods for
determining the order of the nucleotide bases in a DNA fragment
The advent of DNA sequencing has significantly accelerated
biological research and discovery.
The rapid speed of sequencing attainable with modern DNA
sequencing technology has been instrumental in the large-scale
sequencing of the human genome, in the Human Genome Project.