DNA FINGERPRINT

DNA Fingerpriunt
Farmasi Forensik
Prof.Drh.Darmono,M.Sc
Dra.Mayagustina Andarini,M.Sc
DNA FINGERPRINT
-Sidik jari (1930)----- Sidik DNA (1989)
-- Sidik jari dapat diubah dengan di operasi
-Sidik DNA:
- Ada pada semua jaringan
-- Tidak dapat diubah
Struktur DNA:
1.
Struktur molekul:
C (cytosin)
G (guanin)
A (adenin)
T (thymin)
2. Bangunan dasar nukleotida:
- gula/sugar
- desoksiribosa
- kelompok phosphat
- 4 nitrogen dasar berpasangan:
A-T;
C-G;
G-C;
T-A;
Determinasi
@ Segmen DNA dideterminasi dari pasangan gula phosphat
@ DNA membedakan :
- Karakter
- Organisme
- Individu
Nucleotida (asam amino)
Chromosome
Chromosome
3
Kromosom
Diagram of a
replicated and
condensed metaphase
eukaryotic
chromosome. (1)
Chromatid – one of
the two identical parts
of the chromosome
after S phase. (2)
Centromere – the
point where the two
chromatids touch, and
where the
microtubules attach.
(3) Short arm. (4)
Long arm.
2
4
1
KROMOSOM
An image of the 46 chromosomes,
making up the diploid genome of
human male.
Analisis DNA
1.
Isolasi DNA: darah, rambut, kulit, sperma dsb
2.
Memotong mengukur dan mensortir (restriksi):
dengan enzim restriksi asal bakteri:
mis Eco RIpada sequen GAATTC
3.- Elektroforesis gel
- Transfer DNA ke nylon
4. Probing:
-dengan radioaktif- DNA.FP
- Pewarna probDNA.FP
5. DNA. FP dengan pewarna prob lagi
Sidik DNA
Setelah proses elektroporesis, kemudian
diwarnai:
Dengan methylen blue untuk pewarnaan
gel Konvensional
Pewarnaan khusus untuk
peningkatan penampilan dan
memudahkan pembacaan,
dapat diwarnai :
Dengan: - Carolina blue, atau
- Quikviem DNA
Kedua jenis pewarnaan ini
dapat mengurangi
back ground dan meningkatkan
sensitiviti
POLYMERASE CHAIN REACTION (PCR)
Reaksi penggandaan DNA:
Diperlukan:
- DNA original
- dua molekul primer nukleotida utuh
- larutan buffer
- Taq DNA polimerases (enzim):
Fungsi/kegunaanya: - Melipatgandakan DNA dengan cara
mengkopi(copy)
- Diagnosis untuk mengkonformasi DNA
PCR
The polymerase chain reaction (PCR) is a technique in
molecular biology to amplify a single or few copies of a piece
of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence.
The method relies on thermal cycling, consisting of cycles of
repeated heating and cooling of the reaction for DNA melting
and enzymatic replication of the DNA. Primers (short DNA
fragments) containing sequences complementary to the target
region along with a DNA polymerase (after which the method
is named) are key components to enable selective and
repeated amplification. As PCR progresses, the DNA
generated is itself used as a template for replication, setting in
motion a chain reaction in which the DNA template is
exponentially amplified. PCR can be extensively modified to
perform a wide array of genetic manipulations.
Prosedure PCR
•
•
•
•
•
•
Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if
extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for
DNA polymerases that require heat activation by hot-start PCR.[9]
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to
94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen
bonds between complementary bases, yielding single strands of DNA.
Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing
annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is
about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are
only formed when the primer sequence very closely matches the template sequence. The polymerase
binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq
polymerase has its optimum activity temperature at 75–80 °C,[10][11] and commonly a temperature of
72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand
complementary to the DNA template strand by adding dNTPs that are complementary to the template
in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the
end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase
used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum
temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum
conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step,
the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific
DNA fragment.
Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15
minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the
reaction
Proses PCR
Denaturasi
annealing primer
Replikasi DNA
Denaturasi (suhu 94-96oC)
Dobel helix strand dipisah
Annealing Primer (suhu 50-65oC)
annealing/primer melekat pada masing-masing strand
Replikasi DNA (suhu 72oC)
masing-masing strand digandakan
Aplikasi DNA FP
1.
Mendiagnosis Kelainan keturunan (genetik)
- pada janin yang belum dilahirkan
- Bayi yang baru dilahirkan
2.
Penelitian mengenai kelainan genetik
Normalbandingkan --Kelainan
3.
Bukti biologik: untuk laboratorium forensik
Penyakit Genetik
Gen
makhluk
hidup
Lingkungan
Klasifikasi penyakit genetik
Penyakit
Genetik
Autosomal
dominan
Autosomal
resesif
*Achondroplasia
*Huntington
-disease
*Marfan –
disease
*Cystic –
fibrosis
*Sickle cel –
anemia
*Spinalmuscular
atrophy
X-Y-link
X-link
Dominant:
*Aicardi
*Klinefelter
*Hipophospatemi
a
X-link
Resesif:
*Hemophilia
*Buta warna
*Muscular
destrofi
Mitokondri
a
Y-link:
*Infertility
pria
*Leber
hereditary
optic
neurophaty
Multifactorial
*Autisme
*Hipertensi
*Cancer
*Mental –
dis.
dsb
Achondroplasia
ENZIM UNTUK RESTRIKSI
•
Enzim
•
-------------------------------------------------------------------------------------------------------------------------
•
•
•
•
•
•
•
•
•
•
•
•
EcoRI
BamHI
Bgl/II
PvuII
PvuIII
HindIII
HinfI
Sau3A
AluI
TaqI
HaeIII
NotI
Organisme
Escherichia coli
Bacillus amyloliquefaciens
bacillus globigii
proteus vulgaris
Proteus vulgaris
haemophilus influenzaeRd
Haemophilus influenzaeRf
Staphylococcus aureus
Arthrobacter luteus
Thermus aquatus
haemophilus aegyptius
Nocardia otitidis-caviarum
sequen yang
dikenal
GAATTC
GGATCC
AGATCT
CGATCG
CAGCTG
AAGCTT
GANTC
GATC
AGCT
TCGA
GGCC
GCGGCCGC
Mutasi DNA
Kelainan keturunan
Kasus perselingkuhan (hal. 51)
Kasus perkosaan (hal 52)
Kasus perkosaan (hal. 53)
MINI-SATELIT
Variable Number Tandem Repeat (VNTR)
-Pada kromosom manusia ada sequens pendek yang diulang
-Ulangan : dari 1-30 ulangan
-Dapat dipotong pada variasi jumlah tandem repeat: dengan cara
- hibridisasi dan probe spesifik
“TAAGGGCCATAAGGGCCATAAGGGCCA”
VNTR
VNTR
Ayah
Ibu
Ayah
---------------Anak------------------
Ibu
VNTR
Single locus:
-memperlihatkan locus-tunggal probe VNTR sangat mudah
diinterpretasikan untuk setiap individu dengan band yang tidak
lebih dari dua
-Digunakan di Amerika untuk penyidikan forensik dan jejak
keturunan
Multi locus:
-Memperlihatkan locus-multi probe VNTR, banyak informasi
yang dapat dibaca secara simultan dengan band antara 10-30
Tetapi agak susah interpretasinya
-Digunakan di Eropa untuk penyidikan forensik dan jejak
keturunan