Molecular Diagnostics and CRISPR Flashcards
what is a unique feature of the DNA recognition sites used by restriction enzymes?
palindromes - strands of DNA mirror each other in opposite directions
ex: 5’ — AAGCTT — 3’
3’ — TTCGAA — 5’
what buffer is gel electrophoresis soaked in for visualization
ethidium bromide - binds DNA
how does one increase stringency of hybridization assays
increase temperature, add denaturing agent, shorter probe
basically, by making it easier for DNA to denature, you are ensuring that what is left is a really good match because it would have a stronger bond
use higher stringency test to detect very specific nucleotide sequence (differs from probe by just a single or few nucleotides)
what would be the advantage of using a low stringency hybridization assay?
allows for cross-specific analysis and identification of distantly related members of gene family
what is the advantage of converting mRNA to cDNA using reverse transcriptase from retroviruses?
cDNA does not have introns (mRNA does)
for each of these assays, what are you analyzing and what kind of probe do you use?
southern, northern, western blot
southern: DNA sample, cDNA probe
northern: RNA sample, cDNA probe
western: protein sample, antibody probe
what molecular diagnostic method should you use to detect a specific DNA fragment
southern blot
what are ASOs used for
allele-specific oligonucleotides
short piece of DNA (15-20bp) that is complementary to specific sequence
used as hybridization in Dot blot assay (to screen for DNA mutations or polymorphisms)
designed to be specific for one allele of DNA being tested
can detect difference as small as 1 bp
you are looking for an assay to target the mutant allele of a newly found gene in humans. what molecular diagnostic test should you use?
Dot blot assay - screens for DNA mutations or polymorphisms using allele-specific oligonucleotides (ASOs)
this molecular diagnostic method takes advantage of differences in sequences between individuals that can result in different patterns of restriction enzyme digestion. What is?
restriction fragment length polymorphism (RFLP): run DNA on gel, look for restriction enzyme patterns
[differences are not necessarily due to mutations]
you have a mixture of mouse RNA, but you want to detect a specific RNA molecule to measure its expression. what molecular diagnostic technique will you use?
northern blot: RNA sample, cDNA probe
hybridize with a sequence specific probe
combined with electrophoresis (separates by size), northern blots can be used to detect alternatively spliced RNA variants
what is the best molecular diagnostic method to monitor the expression of thousands of genes simultaneously?
DNA microarray
what are the steps in PCR
- denaturation
- annealing (primer binding - each strand needs a primer)
- extension (DNA synthesis)
how is allele-specific PCR (AS-PCR) specific for one allele?
3’ end is specific for one allele and not the other
when utilizing RT-PCR, what is made with the reverse transcriptase?
cDNA from RNA, which is amplified using PCR
what are the disadvantages of northern blots (4)
labor intensive/ time consuming, can probe only 1 gene at a time, need to know the specific sequence to design a probe, and requires a lot of sample
what are the advantages of RT-qPCR (with one caveat)
only need a small amount of sample, can quantify amount of target in sample, faster/easier, can analyze expression of multiple genes at once BUT each needs a specific primer
if you are looking to determine differences in mRNA expression profiles, what molecular diagnostic technique should you use
microarray analysis - not looking for specific gene, can be used for global mRNA expression
why are ddNTPs (dideoxyriboses) useful in DNA sequencing (Sanger method)?
lack the necessary 3’ OH to make the next phosphodiester bond in chain elongation -> serve as chain terminators
what are the 2 things you need (simply speaking) to insert a DNA fragment into bacterial plasmids
restriction enzymes and DNA ligase
plasmid can now be used as a vector for cloning
what is a genomic library
set of bacteria each carrying a different DNA fragment of human genome
cDNA libraries can also be made using mRNA from specific tissues/cell types
to isolate bacterial clone form library with target DNA sequence, do colony hybridization
how are reporter genes engineered to allow for pattern expression of a gene?
replace coding sequence for protein with one for reporter protein (GFP or luciferase, for example)
what is a non viral gene delivery system for gene therapy
liposome
what is unique about engineered packaging cells that allows them to be used for gene therapy?
engineered to express viral proteins required to assemble a vector virus for gene therapy
when would siRNA treatment be a better option that gene replacement therapy?
when you want to turn OFF a gene, such as with an autosomal dominant disorder of inappropriate gene expression in cancer cells
another example: down-regulate PCSK9 (which enhances LDL receptor degradation) to lower cholesterol
what is the general mechanism of the CRISPR system
guide RNA associates with Cas9 nuclease
RNA/Cas bind to complementary sequence in invading viral DNA
Nuclease activity of Cas cuts viral DNA to produce DSB
relies on host cells repair mechanism (risk of off-target Cas binding, and challenging for precise nucleotide changes)
what is the general mechanism of single base editing
retain Cas9 but abolish nuclease activity - no DSB
add cytosine deaminase, which changes C to U
inhibit endogenous BER so U pairs with A and GC—>TA
describe what is different about “prime” editing CRISPR
includes desired edited sequence directly as extension of guide RNA
modified Cas9 to have reverse transcriptase that induces edit right into genome
reduces number of unintended changes
what enzyme is needed to analyze the pattern of gene expression between normal and malignant tissue (need to know the test you should use to answer the question)
RNA microarray would be most useful test - this requires reverse transcriptase
what does an unbalanced base composition of nucleic acid suggest (as in, % thymine does not equal % adenine)
DNA is single stranded - must be from a viral genome since viruses can have ssDNA
what could cause a loss in observable enzyme activity, if there is a normal amount and size of mRNA found?
nonsense mutation - causes premature termination of translation (mRNA would be normal)
which of these tests would you most likely pair with RFLP?
a. ELISA
b. mass spectrometry
c. Northern blot
d. Southern blot
RFLP (restriction fragment length polymorphism) detects DNA polymorphisms
Southern blot can then be used to detect different lengths of DNA fragments
