Transcript DNA – RNA - Protein

Molecular Diagnosis &
Gene Therapy
Asmarinah
Department of Medical Biology
Faculty of Medicine, University of Indonesia
Molecular diagnosis
= nucleid acid-based diagnosis of human disordes
is detection of the various pathogenic mutation in
DNA and/or RNA sample (change in gene expression)
= laboratory medicine + knowledge/technology of
molecular genetics
 benefiting from the discoveries in the field of
molecular biology
Discoveries in
the field
molecular
biology which
influenced the
development of
molecular
diagnostic
Need to know:
DNA – RNA - Protein
Replication
(product: DNA)
Transcription
(product: mRNA)
Translation
(product: protein)
continued
Molecular Dignostic Testing
◘ facilitate:
- the detection and characterization of disease
- the monitoring the drug response
◘ assist in identification:
- genetic modifier: introduced gene which
produce a novel protein that can convert the
trait of interest
- disease susceptibilty
The primary function of molecular diagnostics:
 detection of mutations and single nucleotide
polymophism (SNPs) that are associated with phenotypes
▪ Pauling et al., 1949
 discovered single amino acid change in β-globin
chain sickle cell anemia
 introduced “molecular disease”
term
 set the foundation of molecular
diagnosis
Sickle-cell disease
1300
1100
Mutation
detection:
Methods:
 PCR
 PCR-RFLP
 PCR-ARMS
 PCR-ASO.
 PCR-DNA sequencing
 real time PCR for quantitation
 Microarray
Specimens:
• peripheral blood/cord blood lymphocytes
• villi choriales
• amniotic fluid
• semen, hair
The discovery of PCR method,
(Saiki et al., 1985; Mullis & Faloona, 1987)
 greatly facilitated in principle revolutionized
molecular diagnosis
 foundation for the design and development of
mutation detection, based on amplified DNA
The most
powerfull feature
of PCR:
 getting the
large amount of
copies of the
target sequence,
generated by its
exponential
amplification
PCR-based techniques that can be applied to detect
known point mutation or SNPs in DNA:
1. PCR-ARMS (Amplification Refractory Mutation
System)
 based on the principle that a mismatch between
the 3’ nucleotide of a PCR primer and the template
reduce or prevent primer extention by Taq
polimerase
2. PCR-ASO (Allele-Specific Oligonucleotide)
 based on hybridization of PCR product to allelespecific oligonucleotide probes
Schematic of PCR-ARMS assay for detection of
single base mutation (underlined)
Example:
Detection of DHCR7 gene
mutation that associated
with Smith-Lemli-Opitz
syndrome
PCR-ASO (Allele-Specific Oligonucleotide)
ASO probes is designed to be complementary and
specific for various alleles
Example 
PCR-DNA sequencing
 golden standard and
definitive experimental
procedure for mutation
detection
DNA sequencing result
Mutation in exon 6 of VDAC3 gene in sperm with low motility
174
Sequence of PCR
product from sperm
with normal motility
Posisition 174 : AAG
(Lysine)
Sequence of PCR
product from sperm
with low motility
Posisition 174 : GAG
(glutamic acid)
(Asmarinah et al, 2005)
Real-Time PCR (RT-PCR)
Quantification of the PCR product in real-time,
during the exponential phase of the PCR reaction
One of methods that widely used:
RT-PCR using fluorescent DNA intercalating
dyes (such as SYBR Green 1, ethidium bromide)
The principle:
-The dye incorporates
into groove of dsDNA
-During the PCR
reaction, the amount of
double stranded target
will increased, paralleled
by an increased in SYBR
Green I incorporation
(fluorescent emission)
Quantification of PCR product by RT-PCR method
Amplification plot
Rn = fluoresence detected
at a certain point of
reaction – initial
fluoresence
Ct = threshold cycle
By plotting the Ct value of an unknown sample on the standard
curve  the amount of target sequence in the sample can be
determined (automatically by soft program in RT-PCR maschine)
Present-day mutation detection techniques  for high
througput mutation analysis : DNA Microarray
The principle:
-Oligonucleotides of known sequence are immobilized
onto appropriate surface  DNA chip
-Hybridization of the target to the microarray
-Detection of hybridization, using fluorescent dyes
-Quantification by high-resolution fluorescent scanning
and will be analyzed by computer software
Simultaneous detection of a great nummber of DNA
alteration (genome-wide screening)
Gene Therapy
is a technique for correcting defective genes responsible for
disease development.
Several approaches for correcting faulty genes:
* A normal gene may be inserted into a nonspecific location
within the genome to replace a nonfunctional gene. This
approach is most common.
* An abnormal gene could be swapped for a
normal gene through homologous recombination.
* The abnormal gene could be repaired through
selective reverse mutation, which returns the gene
to its normal function.
* The regulation (the degree to which a gene is
turned on or off) of a particular gene could be
altered.
Methods in gene therapy:
-Using virus as delivering agents:
* Retroviruses, that can create double- stranded DNA copies
of their RNA genomes
* Adenoviruses, viruses with double-stranded DNA genomes
that cause respiratory, intestinal, and eye infections in humans.
* Adeno-associated viruses, small, single-stranded DNA
viruses that can insert their genetic material at a specific site on
chromosome 19.
* Herpes simplex viruses, double-stranded DNA viruses that
infect a particular cell type, neurons. Herpes simplex virus type
1 is a common human pathogen that causes cold sores.
•Non-viral methods
- Sythetic oligonucleotides
to inactivate the genes involved in the disease process,
with:
antisense specific to the target gene to disrupt the
transcription of the faulty gene.
siRNA (small/short interfering or silencing RNA)
to signal the cell to cleave specific unique
sequences in the mRNA transcript of the faulty
gene, disrupting translation of the faulty mRNA,
and therefore expression of the gene
-Liposome,
an artificial lipid sphere with an aqueous core,
which carries the therapeutic DNA and capable of passing
the DNA through the target cell's membrane.
Obstacle factors for gene theraphy becoming an effective
treatment for genetic diseases:
* Short-lived nature of gene therapy
* Immune response
* Problems with viral vectors
* Multigene disorders
References:
•Alberts et al., 2002. Molecular Biology of the Cell. 4th ed.
•Karp, 2005. Cell and Molecular Biology. 4th ed.
•Patrinos & Ansorge, 2006. Molecular Diagnostics.
•http://www.ornl.gov/sci/techresources/Human_Genome/
medicine/genetherapy.shtml (16 April 2009 jam 15.00
WIB)