Molecular Diagnostics and CRISPR

Question 1

what is a unique feature of the DNA recognition sites used by restriction enzymes?

Answer 1

palindromes - strands of DNA mirror each other in opposite directions

ex: 5’ — AAGCTT — 3’
3’ — TTCGAA — 5’

Question 2

what buffer is gel electrophoresis soaked in for visualization

Answer 2

ethidium bromide - binds DNA

Question 3

how does one increase stringency of hybridization assays

Answer 3

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)

Question 4

what would be the advantage of using a low stringency hybridization assay?

Answer 4

allows for cross-specific analysis and identification of distantly related members of gene family

Question 5

what is the advantage of converting mRNA to cDNA using reverse transcriptase from retroviruses?

Answer 5

cDNA does not have introns (mRNA does)

Question 6

for each of these assays, what are you analyzing and what kind of probe do you use?
southern, northern, western blot

Answer 6

southern: DNA sample, cDNA probe

northern: RNA sample, cDNA probe

western: protein sample, antibody probe

Question 7

what molecular diagnostic method should you use to detect a specific DNA fragment

Answer 7

southern blot

Question 8

what are ASOs used for

Answer 8

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

Question 9

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?

Answer 9

Dot blot assay - screens for DNA mutations or polymorphisms using allele-specific oligonucleotides (ASOs)

Question 10

this molecular diagnostic method takes advantage of differences in sequences between individuals that can result in different patterns of restriction enzyme digestion. What is?

Answer 10

restriction fragment length polymorphism (RFLP): run DNA on gel, look for restriction enzyme patterns

[differences are not necessarily due to mutations]

Question 11

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?

Answer 11

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

Question 12

what is the best molecular diagnostic method to monitor the expression of thousands of genes simultaneously?

Answer 12

DNA microarray

Question 13

what are the steps in PCR

Answer 13

1. denaturation
2. annealing (primer binding - each strand needs a primer)
3. extension (DNA synthesis)

Question 14

how is allele-specific PCR (AS-PCR) specific for one allele?

Answer 14

3’ end is specific for one allele and not the other

Question 15

when utilizing RT-PCR, what is made with the reverse transcriptase?

Answer 15

cDNA from RNA, which is amplified using PCR

Question 16

what are the disadvantages of northern blots (4)

Answer 16

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

Question 17

what are the advantages of RT-qPCR (with one caveat)

Answer 17

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

Question 18

if you are looking to determine differences in mRNA expression profiles, what molecular diagnostic technique should you use

Answer 18

microarray analysis - not looking for specific gene, can be used for global mRNA expression

Question 19

why are ddNTPs (dideoxyriboses) useful in DNA sequencing (Sanger method)?

Answer 19

lack the necessary 3’ OH to make the next phosphodiester bond in chain elongation -> serve as chain terminators

Question 20

what are the 2 things you need (simply speaking) to insert a DNA fragment into bacterial plasmids

Answer 20

restriction enzymes and DNA ligase

plasmid can now be used as a vector for cloning

Question 21

what is a genomic library

Answer 21

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

Question 22

how are reporter genes engineered to allow for pattern expression of a gene?

Answer 22

replace coding sequence for protein with one for reporter protein (GFP or luciferase, for example)

Question 23

what is a non viral gene delivery system for gene therapy

Answer 23

liposome

Question 24

what is unique about engineered packaging cells that allows them to be used for gene therapy?

Answer 24

engineered to express viral proteins required to assemble a vector virus for gene therapy

Question 25

when would siRNA treatment be a better option that gene replacement therapy?

Answer 25

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

Question 26

what is the general mechanism of the CRISPR system

Answer 26

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)

Question 27

what is the general mechanism of single base editing

Answer 27

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

Question 28

describe what is different about “prime” editing CRISPR

Answer 28

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

Question 29

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)

Answer 29

RNA microarray would be most useful test - this requires reverse transcriptase

Question 30

what does an unbalanced base composition of nucleic acid suggest (as in, % thymine does not equal % adenine)

Answer 30

DNA is single stranded - must be from a viral genome since viruses can have ssDNA

Question 31

what could cause a loss in observable enzyme activity, if there is a normal amount and size of mRNA found?

Answer 31

nonsense mutation - causes premature termination of translation (mRNA would be normal)

Question 32

which of these tests would you most likely pair with RFLP?
a. ELISA
b. mass spectrometry
c. Northern blot
d. Southern blot

Answer 32

RFLP (restriction fragment length polymorphism) detects DNA polymorphisms

Southern blot can then be used to detect different lengths of DNA fragments