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Lecture 49: Applications in Molecular Genetics II- Known & New…
Lecture 49: Applications in Molecular Genetics II- Known & New Mutations
Detecting KNOWN Mutations
(
adding to lec 48)
DNA/Genotyping Microarrays
:red_flag:
Reverse transcribe
mRNA to cDNA
Label w/
fluorescent dyes
Hybridize
to special
exon
arrays
Compare relative signal to derive relative
mRNA quantity
Red
= HIGH expression of gene
Green
= LOW expression of gene
Application
Advantages:
Automated,
high
-throughput analysis of specific mutations or polymorphisms
can study
several
samples for several pt.'s at same time
less hands-on work/sample
automation- data interpreation
Disadvantages:
expensive $$$
NOT suitable for
low
-volume testing
(
use & throw)
Flow Cytometry
:explode:
Protein-based method
Based on
antibody-based fluorescent tagging
Analysis: 1 cell at a time!
by focusing laser &
measuring light scatter & intensity of fluorescence
Applications
Leukemia
Fetal RBCs in mother's blood
CD4+ cell count- HIV
Blood transfusions
Cancer detection
Pre/post transplant
ELISA
:explode:
DIRECT
ELISA :fire:
Attachment of antigen to polystyrene plate followed by enzyme-labeled antibody that can react w/antigen & substrate that can be measured
INDIRECT
ELISA :fire:
Attachment of antigen to polystyrene plate -> unlabeled OR primary antibody -> enzyme-labeled antibody that can react w/BOTH primary antibody & substrate
"Immunologic technique widely used for Antigen/Antibody detection in clinical samples"
Applications
:
-Hormones
-Allergens
-Viral + bacterial antigens
-Antibodies (in response to vaccine/infection)
PCR Applications
Genetic variations
Detection
of
Rare sequences
Digital PCR (dPCR)
Real-time quantitative PCR (qPCR)
Forensics & Paternity Testing
DNA Fingerprinting
(PCR in Forensics) :silhouettes:
Highly polymorphic hypervariable repeat regions exist in genome
Short Tandem Repeats (STR's)
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Variable Number Tandem Repeats (VNTRs)
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used to
distinguish 1 person from another!
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Residual disease- chemotherapy pt.'s and/or hem. cell transplant. for hema malignancies
COVID-19 diagnosis!
Antibiotic-resistance genes
Viral load (HIV, EBV, H1N1)
Rare allele detection
Residual disease in patients receiving chemotherapy and/or hematopoietic cell transplantation for hematologic maligncies
CNVs
HSV in Kaposi's sarcoma
Preimplantation testing & non-invasive prenatal testing (NIPT)
SNP genotyping
microsatellites indels, CNVs
Detecting NEW Mutations
Blotting
:checkered_flag:
Southern Blotting
(DNA)
DNA
sample separated based on SIZE using
gel electrophoresis
--> transferred to
nitrocellulose/nylon membrane
radioactive DNA probe binds to complementary sequences
Applications/Advantages:
case-by-case basis
large deletions, duplications, gene rearrangements
Trinucleotide expansion
Methylation/deletion of SNRPN locus in PWS/AW*
Her+ Breast cancer
Nonradioactive methods-possible
Disadvantages:
Labor + Time-intensive
Not good quantitative
Need large DNA amounts
Northern Blotting
(RNA)
RNA
= target
Western Blotting
(Protein)
PROTEIN
= target
Applications:
Bovine spongiform encephalopathy
HIV
Lyme disease
NEWER technique=
Immunohistochemistry
In native tissue samples
(i.e., tumor histopathologies)
Southwestern blotting
DNA-binding proteins
Serum Protein Electrophoresis (SPEP)
followed by Coomassie staining
(1) Sanger Sequencing
3'-OH allows strand extension at 3'end
chain-termining
ddNTP
ddNTP's
are
derivates
of normal dNTP that
LACK 3'-OH group
when put into growing DNA strand, they
block
further elongation
Automated Sanger Sequencing Electrophoresis
Applications:
precise identifications of ALL DNA variations
both known + unknown mutations
(2) Next Generation Sequencing (NGS)
:fire:
Parallel sequencing of millions of reads on single chip
Whole genome, exome & targeted gene parels
Validation- Sanger sequencing (
more accurate
)
LOW cost + HIGH speed
Personalized/precision medicine
Relies on massively
parallel sequencing
reactions out on clusters of
PCR-amplified DNA
NGS Applications
when
large #
of pathogenic genes need to be screened
Diagnosis-complex diseases
Cancer screening + management
Infections diagnosis
Healthy people
Biomedical research
Obvious candidate genes
Cost LESS than sequencing indv. genes
GENE MODIFICATION STRATEGIES
:red_flag:
2. Gene Knock-Out/Knock-In
:star:
KEY:
Cre-mediated recombination
and
loxP sequence
global/targeted
spatial/temporal
"Knock-IN" method
mutated DNA seq. exhanged for endogenous seq. w/out any disruption of gene
gene for
Cre-recombinase
knocked
into
targeted loci --> brings expression under endogenous gene promoter
allows
tissue-specific
or
temporal-specific
expression of
Cre enzyme
end: recombination of
loxP
sites that flank gene of interest
3. Gene Knockdown/Silencing
RNA Interference :star:
KEY:
Dicer, RISC Complex
dsRNA
synthesized=
complementary
to
mRNA
interest sequence
When exposed to cells/tissues,
dsRNA separates
&
promotes
degradation
of
target mRNA
,
knocking DOWN gene expression
4. Genome Editing
:star:
(CRISPR/Cas9)
CRISPR/Cas9= gene-editing tool derived from bacterial defenses against viruses & foreign plasmids
2 Key Components
guide RNA (gRNA)
binds
complementary
to target DNA sequence
2. Cas9
endonuclease
that causes
dsDNA break
that requires repair
Steps
:
Target sequence
Guide RNA binds to target sequence
Cas9 Enzyme binds to guide RNA
Cas9 "cuts" both DNA strands
Cut repaired -> introducing mutation!
is
creation of sequence-specific alternations in DNA using mol methods that take advantage of
site-directed DNA repair
after strand breakage
used to correct pathogenic gene variant or alter other biologic processes
investigational method
1. GENE THERAPY
:star:
Introduction of
exogenous
genes into 1+
autologous
/
allogenic
cell types
Gene Therapy Vectors
Some FDA-Approved Drugs
Kymriah
B-cell precursor ALL
B-cell lymphoma
Lentivirus
Yescarta
relapsed or refractory large B-cell lymphoma
retrovirus
Imlygic
unresectable lesions in melanoma
live, attenuated herpes simplex virus
Luxturna
inherited form of retinal dystrophy
adeno-associated virus
CHALLENGES
Unwanted immune system Rxn
Targeting wrong cells
Infection by virus
Tumor possibility
cost
Antibody-Based Technologies
DNA Sequencing- 2 Major Types
