Molecular Diagnostics (exam 2) Flashcards
3 methods of amplification
target amplification systems
probe amplification systems
signal amplification
target amplification
copy directly from the target (RNA/DNA)
probe amplification systems
not copying off the organism, but something else initially bound to the target is being copied
signal amplification
can see if something is amplified or not; amount of signaling
Target amplification methods
PCR
TMA - transcription mediated
SDA - strand displacement
Types of PCR
PCR with specific probes
Nested PCR
Multiplex PCR
Nested PCR
use two sets of amplification primers, one internal to the other
start off wide, narrow in on a region
Multiplex PCR
two or more sets of primers specific for different targets
multiple PCR reactions at once
signal amplification example
bDNA - branched DNA probes
probe amplification example
LCR - ligase chain reaction
how many targeted sequences are produced?
10^8 - 10^9 copies
How did Kary Mullis invent PCR?
knew that template DNA could be exposed
knew that primers initiate DNA synthesis
Knew that cheap, commercial enzyme was available
Mullis wanted a way for PCR to
generate large amounts of DNA from a single copy
Steps of PCR
- denaturation
- annealing of primers
- extension of new strand
what is needed for a PCR reaction?
primers
thermostable polymerase
target nucleic acid (RNA/DNA)
primers may be
specific or random
what can dictate primer length?
annealing temperature and specificity
PCR primers
ss 18-30 b DNA fragments complementary to sequences flanking the region to be amplified
the larger the distance between the primers,
the larger the size of the PCR product
Tm
for short (14-20 bp) oligomers
= 4 (GC) + 2 (AT)
Most common thermostable polymerase used and the source it came from
Taq polymerase
from T. aquaticus
a reaction mix contains
all the components necessary for DNA synthesis
what is in a standard PCR reaction mix?
primer
nucleotides (dNTPs)
KCl
Tris buffer
MgCl2
polymerase
copies of template
Temperatures at each step of PCR
denaturation - 90-96, 20 seconds
annealing - 40 -68, 20 seconds
extension - 70-75, 30 seconds
for Primer design,
avoid inter-strand homologies
avoid intra-strand homologies
Tm forward primer = Tm reverse primer
avoid longer than GGGG
product size - 100bp-700bp
target specificity
for long primers in PCR,
94-60-72
for adequate primers in PCR,
94-50,72
for uncontrolled results of PCR,
94-37-65
the amplification program consists of a series of
20-50 PCR cycles
1st major advancement of PCR
thermostable polymerase
2nd major advancement of PCR
automation
why are thermostable polymerases used in PCR?
because they can withstand repeated high denaturation temperatures
3rd advancement in PCR
detection
gel electrophoresis –> micrometers –> microassays
microarrays can
detect thousands of samples
the most dangerous contamination is
PCR product from a previous reaction
what is a potential source of contamination
any molecule of DNA containing the intended target sequence
most common cause of contamination
carelessness and bad technique
ways to reduce contamination
separate pre and post PCR labs
dUTP and uracil-N-glycosylase
Psoralen and UV
10% bleach
dUTP and uracil-N-glycosylase
chews up DNA with uracil in it
degrades contaminants from previous PCR reactions
controls for PCR and what they control
blank - contamination
negative - specificity
positive - sensitivity
blank reaction
all reagents except DNA template
negative control reaction
all reagents and a DNA template lacking the target sequence
positive control reaction
all reagents and a known target containing DNA template
COBAS Amplicor Analyzer
samples are amplified and products detected automatically after the PCR reaction
Real time/quantitative PCR
products are detected by fluorescence during the PCR reaction
allows for quantification of starting material
4th advancement of PCR and example
combining amplification and detection
ex: COBAS amplicor
5th advancement of PCR and example
combining everything!
ex: COBAS 6800/8800
the length of the lag phase in qPCR is
inversely proportional to the amount of starting material
difference between end point PCR and real time PCR
end point - simple +/- results
real time - complex results
hybridization analysis
probe hybridization
only bind and fluoresce the intended PCR product
HIV case study led to
a perfect match between PCR and immunodiagnostic product
PCR is much faster, more efficient
Lyme disease case study led to
PCR being able to detect differences between types of infections (new vs recurrent)
Sars CoV2 case study led to
PCR having great results in sensitivity and it is very specific so no false positives
advantages of PCR
specific
simple and fast
can amplify low quantities
works on damaged DNA
sensitive
flexible
limitations of PCR
contamination risk
primer complexities
detection methods
primer-binding site complexities
transcription amplification method
isothermal RNA amplification modeled after retroviral replication
transcription amplification method process
RNA is reverse transcribed into cDNA followed by RNA synthesis via RNA polymerase
primer contains the T7 RNA pol promoter sequence
3 enzymes in TMA
reverse transcriptase
T7 RNA pol
RNAse H
Hologic Analyzer
shuffles through RNA and DNA
TMA approach
strand displacement amplification
isothermal
enzyme comes in, cuts and then pushes the strand out of the way
ligase chain reaction
probe amplification
probes bind immediately adjacent to one another on a template
bound probes are ligates and become templates for the binding of more probes
branched DNA detection
captures probe hybrid
2nd probe binds to first probe
hybridize bDNA amplifier
addition of alkaline phosphate molecules
RFLP analysis
Restriction fragment length polymorphism
use of restriction enzymes to recognize specific DNA sequences and cut them
RFLP means that there are
polymorphisms between people in the number of restriction sites
issues with RFLP analysis
can not detect all mutations - it has to coincide with a RE cut site
genetic testing examples
predictive testing
newborn screening
carrier testing
predictive testing
carries a mutation that will cause or put them at a higher risk for disease later in life
newborn screening
detects common disorders in newborns
carrier testing
carries a mutation that could be passed on to offspring
Thalassemia
blood related genetic disorder
involves errors in hemoglobin genes
most common inherited single gene disorders
alpha and beta thalassemias
CRISPR
DNA editing technique
CRISPR gene therapy for sickle cell anemia
- harvest bone marrow stem cells
- Cas9 guides RNA
- CRISPR/Cas9 genome editing
- implant edited stem cells in patient
hemophilia
partial/total lack of blood clotting factor
can be diagnosed in infants after 9 months
hemophilia A vs hemophilia B
A - deficiency in factor 8
B - deficiency in factor 9
Cystic fibrosis
affects respiratory, digestive and reproductive systems
thick mucous linings produced in the lungs
CFTR gene contains
27 exons, 250 kb of DNA
how many mutations in the CFTR gene result in CF disease?
over 200
the most common mutation in CFTR gene causing CF
3 base pair deletion at codon 508
loss of phenylalanine residue blocks transport of CFTR protein
what is used to diagnose cystic fibrosis?
dot blot
dot blot
DNA from a patient is spotted out twice
one used with a probe complementary to the normal sequence
other with a probe complementary to a mutated sequence
what if someone is a carrier for CF? how will their dot blot look?
faint dots in both normal and mutant probe
Pulmozyme
therapeutic used for CF patients
Tay-Sachs
autosomal recessive disorder fatal genetic disorder
mutation in the HEXA gene on chromosome 15
is there a cure for Tay-Sachs? what has been done to decrease the number of children born with it?
no cure
carrier screening and genetic counseling
characteristics of Tay Sachs at birth?
appear normal
characteristics of Tay Sachs at 6 months?
development slows
characteristics of Tay Sachs at 2 years?
seizures and fading mental functions
characteristics of Tay Sachs at 3 years?
blindness, mental retardation, paralysis, and non responsiveness
detection methods for tay-sachs
amniocentesis
chorionic villus sampling
blood samples to detect carriers
Tay-Sachs disease is the inability to
synthesize an enzyme that prevents lipid build up in brain cells
Tay-Sachs causes
the breakdown of the nervous system
Parent help for Tay-Sachs
genetic counseling and support groups
Fragile X is the
most common cause of inherited mental retardation
Fragile X
loss of function mutations in the fragile X mental retardation 1, FMR1 gene
neurodegenerative, trinucleotide repeat disorder
repeats cause high methylation, blocks transcription
Huntington Disease
autosomal dominant genetic disorder
neurodegenerative - loss of ability to walk, talk, etc.
over 36 CAG repeat in long polyglutamine tract
is there a cure for Huntington disease?
no treatment or cure
examples of polygenic genetic disorders
cancer
diabetes
heart disease
hypertension
obesity
infertility
asthma
Mitochondrial disorders
heart failure, autism, Alzheimers, etc.
caused by mitochondrial genome and not nuclear genome
Next-Gen sequencing
computer aligns sequence at the end, can get the whole genome
high powered sequencing
Why are pharmacists interested in DNA sequencing?
personalized medicine
personalized medicine
tailoring treatments to specific characteristics of each patient
Herceptin (Trastuzumab)
humanized monoclonal antibody designed to target the HER2 receptor domain
will Trastuzumab work for breast cancers that aren’t HER-2 positive tumors?
NO!
only for the specific mutation
