Blok Biologi Molekuler 2018

TEKNIK BIOLOGI
MOLEKULER
Erika Diana Risanti
Bagian Histologi dan Biologi Molekuler FK UMS
Outline
 Isolasi DNA
 PCR
 RFLP
 Sequencing
 IHC (Immunohistochemistry)
DNA Isolation/DNA extraction
DNA isolation/DNA extraction
 A routine procedure to collect DNA for subsequent
molecular or forensic analysis.
 todetect bacteria and viruses in the environment
 diagnosing disease and genetic disorders.

 Paternity test
Sample source for DNA extraction

Swab mukosa Jaringan FFPE

Cairan tubuh Sel kultur Darah

Sample source for DNA extraction

Nucleated cell
 Blood  Tissue
 Whole blood   Fresh
max.24 hours, 4°C  FFPE (Formalin-Fixed
 Frozen Paraffin Embeded)
 In lysis buffer
Principles of DNA extraction

Getting the cell

Membrane cell lysis

Cellular Protein Degradation

*Protein Precipitation *DNA precipitation

Dissolve DNA with TE buffer or pure water

Getting the cells

Blood Tissue FFPE Cell

culture

Separate white
blood cells and
red blood cells Deparafinization Harvest and
trough Homogenization using xylene and resuspend the
ethanol pellet
centrifugation 
buffy coat
Membrane Cell Lysis
 The cell membrane can be lysed by:
 Detergent (SDS)  lipid
 Proteinase K  protein

 To lyse the cell and nuclear membrane  DNA

released
 DNA is soluble in water
Cellular Protein Degradation
 To degrade the protein membrane & cellular
protein (e.g Histon)  to open the DNA complex
structure of chromosome.
 Reagent for protein degradation: Proteinase K
Protein precipitation
DNA precipitation
 Protein precipitation  precipitated the proteins
with sodium or ammonium acetate, or extracted
them with a phenol-chloroform
 DNA precipitation  DNA in the aqueous solution
added by Cold ethanol or isopropanol  DNA
insoluble in ethanol  DNA precipitation

Dissolved DNA
 DNA can be resolubilized in a slightly alkaline
buffer (TE pH 8) or in ultra-pure water
DNA Quality Control
 Optical density measurement  DNA
concentration (quantity)
 spectrophotometer
 Nanodrop spectrophotometer
 QubitTM HS Fluorometer
 DNA purity:
 OD 260/280 ratio = 1.8 – 2 (to check protein contamination)
 OD 260/230 ratio = >1.8 (to check salt, rest of phenol
contamination)
 DNA integrity  electrophoresis (quality)
DNA quantity equipments
DNA integrity using electrophoresis
DNA (negatively charged) migrates based on
fragment size from negative to positive charge
DNA integrity
PCR
Polymerase Chain Reaction
PCR
 Biochemistry and molecular biology technique
 Exponentially amplifying DNA, enzymatic
replication in vitro
 Used in medical and biological research:
 Detection of hereditary disease
 Genetic fingerprints

 Diagnosis of infectious disease

 Cloning of genes

 Paternity testing
 How do we identify and detect a particular
sequence in the genome (certain gene)?

 FACTS!!
 Huge number of bases in the
genome (3x109)  need to
be specified
 DNA samples are usually
limited  need to be
amplified

PCR was invented by DR Kary Murris (1983), who won Nobel price in 1993
PCR Requirements
(all mix in one tube)

1. DNA template  DNA contains gene of interest

2. Primers: forward and reverse
3. Taq DNA polymerase
4. Deoxynucleotides tri phosphats (dNTPs)  dATP,
dTTP, dCTP, dGTP
5. PCR Buffer and MgCl2 (cofactor enzim)
MgCl2 required for the activity of Taq DNA polymerase
Too low conc. : insuficient Taq Pol ac+vity
Too high conc.: less specific PCR
Primers
 Oligonucleotide (2 primers: forward and reverse) that are
complementary to the sense and anti-sense strand of the DNA
target.

 Primers are usually short (15-30 bases). They are hybridized

to a target DNA, which is then copied by the polymerase.
Taq DNA polymerase
 An enzyme originally isolated from the bacterium Thermus
aquaticus and performes as a heat‐stable DNA
polymerase (72-75C).
 Taq pol enzymatically assembles a new DNA strand from
nucleotides bases
 Taq pol from Thermus aquaticus does not have proof-
reading ability
Thermal Cycler
 Heats and cools the reaction tubes to achieve the
temperatures required at each step of the reaction
Steps of PCR
PCR cycle
Denaturating temperature and time
 To separate double stranded DNA (ds DNA)
 Denaturing temperature 91-97°C
 Taq polymerase half-life of 30 min at 95°C
 Cyclenot more than 30
 Reduce denaturation temp, increase the cycle

 Normally 1 min at 94°C

 Or 96°C for 15 second

Annealing temperature
 Depends on:
 primerlength, longer >> temp >>
 Sequence, >> G+C content, >> temp

Tm = 4 (G+C)+2(A+T) °C
 Ta 50°C below the lowest Tm
 Too low Ta, anneal to sequence other than the true
target (non-specific target)
 Too high Ta, too little product will be made

 Anneal efficiently in 30 sec or less

Annealing temperature
Elongation temperature and time
 Normally 70 – 72°C for 0.5 – 3 mins
 Elongation occurs from the moment of annealing
 At around 70°C, primer extension occurs at up to
100 bases/sec
 1 min is sufficient for amplification of 2kb sequence
PCR will be run ....
In theory, PCR reactions
amplify exponentially,
doubling every cycle
In reality, exponential
amplification was
hardly happen

1. An inactivation of the
DNA polymerase
2. A decrease in
concentration of the
primers or the dNTPs
BCR/ABL fusion
 The BCR/ABL fusion
turns out to be positive
in:
 90-95 % of CML
(Chronic Myelogenous
Leukemia) patients
 25 % of adult ALL
(Acute Lymphocytic
Leukemia) patients
 5 % of child ALL (Acute
Lymphocytic Leukemia)
PCR detection for Leukemia diagnosis
RFLP
Restriction Fragment Length Polymorphisms
What is RFLP?
 A technique that exploits variation in DNA
sequence by cut up DNA into fragments using
restriction enzymes.
 A combination of PCR – restriction method to
detect SNP (single nucleotide polymorphsim)
Restriction enzymes
 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
 More than 200 restriction enzymes are commercially
available
EcoRI
 E = genus Escherichia
 co = species coli
 R = strain RY13
 I = first endonuclease identified
Each enzyme have their own rule
 5' overhangs  sticky end

 3' overhangs  sticky end

 Blunts  blunt end

RFLP Principle
RFLP in MTHFR gene
SEQUENCING
DNA Sequencing
 The term DNA sequencing encompasses
biochemical methods for determining the order
of the nucleotide bases in a DNA fragment
 Human Genome Project

 DNA sequencing methods:

 Maxam-Gilbert method
 Chain Termination method (Sanger method)
DNA Sequencing
Maxam-Gilbert Method
 based on chemical
modification of DNA and
subsequent cleavage at
specific bases
 advantage: purified DNA
can be used directly
 disadvantages: complex,
use of hazardous
chemicals, difficulties with
scaling up.
Sanger Method
 Chain termination
(dideoxy) method
 advantage: more efficient,
use fewer toxic chemicals,
use lower amounts of
radioactivity
 disadvantage: required
that each read start be
cloned for production of
single-stranded DNA
Sanger Method
 based on the use of dideoxynucleotides (ddNTPs) in
addition to the normal nucleotides (NTPs) foud in
DNA.
 ddNTPs : essentially the same as nucleotides except
they contain a hydrogen group on the 3’ carbon
instead of a hydroxyl group (OH).
 These modified nucleotides, when integrated into a
sequence, prevent the addition of further nucleotides.
(Speed, 1992) a phosphodiester bond cannot form
between the dideoxynucleotide and the next incoming
nucleotide, and thus the DNA chain is terminated.
Mixture of Sequencing
Each of the four
dideoxynucleotides fluoresces a
different color when illuminated
by a laser beam and an
automatic scanner provides a
printout of the sequence.
IHC
Immunohistochemistry
Imunohistokimia (IHK)
IHC
 Immunohistochemistry (IHC) is a useful research tool
and used to localize specific antigens in tissue
sections with labeled antibodies based on antigen-
antibody interactions.
 The immune reactive products can be visualized by
a marker including fluorescent dye, enzyme in
general; radioactive element or colloidal gold also
can be used.
Contoh IHC, deteksi PCNA pada esofagus
IHC principle
IHC pada Breast Cancer
Alhamdulillah
edr123@ums.ac.id