Which is required to generate cDNA
A. DNA polymerase
B. RNase H
C. Restriction Enzyme
D. Helicase
B
What is the function of Taq
... [Show More] Polymerase
recognize dsDNA
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01:33
You have a 50 μL primer stock at 9pM/μl and want dilute the stock to 900nM. How much stock should you add(what is the dilution factor)
2
What gene causes hypersensitivity to Irinotecan?
UGT1A
Which allele causes hypersensitivity to Carbamazepine?
HLA-b*1502
What gives MLPA its specificity
Stuffer Sequence
How is copy number determined?
MLPA
What is the gene that cause resistance to Vancomycin
vanA
Which assay is FISH best used for
Prader Willi
RNA polymerase II: where is it and what does it do
Eukaryotic: It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. (transcribes mRNA)
Hazard that is blue and is level 4 what does it mean
big health hazard
Dr calls wanting test results but results have not been signed out by a medical director what do you do
don't give out the result until it has been signed out my the MD
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01:33
What trinucleotide repeat is involved in Fragile X?
CGG
In Fragile X, what is considered a full mutation expansion?
200+
What method is used to detect Fragile X?
Southern blot (now PCR /mPCR)
Which gene is the largest and has deletions/duplications as the most common mutation types
DMD
Which HCV geneotype(s) has/have the best prognosis
Genotypes 2 &3
Positive attributes of bDNA
Can be used for both DNA and RNA
What type of primer set is best?
forward primer should complement DNA strand and reverse primer should mirror and inversely complement the forward primer
What are some things that stabilize RNA?
Inactivated and minimizing exposure to environmental/endogenous RNases
mRNA: 5'cap
What is the concentration of DNA with an OD of 2.0
100 μg/mL
Given the following data what can quantifiably be said about DNA when 260 = 1.4 and 280 = 1.1
There is 70ng of pure DNA
Sample is contaminated with proteins and phenols
What is the equation for PCR efficiency
(1+Σ)^n
You have a gel where the positive control worked, the negative control worked, patient 1 had a double band, patient 2 was positive, and patient 3 had no bands. What would you do?
repeat patient 3
How do you detect for methylation
Bisulfate sequencing
MSI (Micro-Satellite Instability) tests for what?
Mismatch Repair Genes
Translocation at t(15, 17)
Translation at t(15, 17)
Coumadin can be affect by a mutation in what gene
CYP2C19-KVORC1
What virus interfere with kidney transplant patients
BK
You have 2 HIV viral load tests 5 days apart 1 being high on day one and 1 being low by day 5. What do you do?
Look into storage and transport
You are given a strand of primers. Calculate the Tm
2˚(AT) + 4˚(GC)
When doing a multiplex you observe faint bands. What do you do
Adjust the annealing temp
What do you do when you want to optimize a PCR reaction that generates short PCR fragments?
Adjust annealing temperature
Which is caused by a T-cell rearrangement
- Mantle Cell
- Burkits
- Hodkins Lymphoma
- Sezary
Sezary
What protects DNA as it is being replicated
Single Stranded Binding Protein
What opens the DNA double helix up
Helicase
What unwinds spiral DNA
topoisomerase
What does HCV assays target
5' UTR region
How does TaqMan work?
fluorescence is generated when the quencher is hydrolyzed
Which is the best order to limit contamination
- Mastermix/ patient/ positive/ negative
- Patient/ Mastermix/ negative/positive
- Matermix/ negative/ patient/ positive
- Mastermix/ positive/ patient/ negative
Matermix/ negative/ patient/ positive
How does Luminex work
Tests one patient with many beads
How do histones bind to DNA
ionic charges
Histones are associated with what amino acids
Lysine & Arginine
Methylation and Histone Modification are examples of what
epigenetics
Variations in Maternal & Paternal DNA mean what
imprinting
HLA typing over serology has the advantage of detecting what
null mutations
Doctor suspects that a patient has clonality you run a PCR and gel and the patient sample, the positive control, and the negative control all show a band. What do you do
repeat testing
How do you remove RNases from glassware
treat with DECP for 4 hours
Which strain of HPV is pathogenic
16, 18
What medication is used to tumor with a mutation in HER2/neu
Herceptin
What gene causes the majority of cancers
p53
What is the mutation for Fac V Leiden
1691G>A
What does Fac V Leiden cause
Deep Vein Thrombosis
What is it called when 2 double stands of DNA is covalently linked by the 5' to 3' ends
ligation
Capillary Electrophoresis uses what
Polyacrylimide
In RFLP wildtype shows 2 bands and a homozygous mutation shows 1 band. What does a heterozygous mutation look like
3 bands
You have two primers with Tm's 40˚ and 51˚. How do you make them work
add bases to the primer with the lower Tm*
You have a fragment 150-300 bp How much agarose do you use
3%
Denaturing HPLC detects what
SNPs (also base substitutions, small deletions, and insertions)
What is the conformation of DNA in its natural state
bDNA right-handed helix
Most common CF mutation
3 nucleotide deletion (F508del)
Which Herpes virus can lead to PTLD (post-transplant lymphoproliferative disorder)
- CMV
- HSV
- EV
- EBV
EBV
RT-PCR detects a gene fusion in the positive, negative and the patient sample what should be done
- Repeat RT-PCR
- Repeat extraction and RT-PCR
- Report out
- Perform Southern Blot
Repeat RT-PCR
What is the equation for PPV(positive predictive value)
PPV=TP/(TP+FP)
(#true positives)/(# true positives + # false positives)
What can you expect from a multiplex PCR
you will need to increase the amount of magnesium
Translation t(14, 18)
Follicular Lymphoma
DNA Methylation will result in which of the following:
- inhibition of transcription
- inhibition of translation
- gene mutation
inhibition of transcription (protein inactivation)
Real-Time PCR is better as a quantitative then conventional tests based on which of the following?:
- Gives you greater dynamic range
- Can monitor each cycle better
- Can use CT to determine results
Can use CT to determine results
For PCR, I get (many) longer unspecific products. What can
I do?
Decrease annealing time
Increase annealing temperature
Decrease extension time
Decrease extension temperature to 62-68º C
Increase KCl (buffer) concentration to 1.2x-2x, but keep
MgCl2 concentration at 1.5-2mM.
Increase MgCl2 concentration up to 3-4.5 mM but keep
dNTP concentration constant.
Take less primer
Take less DNA template
Take less Taq polymerase
If none of the above works: check the primer for
repetitive sequences (BLAST align the sequence with the
databases) and change the primer(s)
Combine some/all of the above
For PCR, I get (many) shorter unspecific products. What can
I do?
Increase annealing temperature
Increase annealing time
Increase extension time
Increase extension temperature to 74-78º C
Decrease KCl (buffer) concentration to 0.7-0.8x, but keep
MgCl2 concentration at 1.5-2mM
Increase MgCl2 concentration up to 3-4.5 mM but keep
dNTP concentration constant
Take less primer
Take less DNA template
Take less Taq polymerase
If none of the above works: check the primer for
repetitive sequences (BLAST align the sequence with the
databases) and change the primer(s)
Combine some/all of the above
My PCR reaction was working before, but now I am not getting any product. What is a possible explanation and what can I do?
Make sure all PCR ingredients are taken in the reaction
(buffer, template, Taq, etc)
Change the dNTP solution (very sensitive to cycles of
thawing and freezing, especially in multiplex PCR)
If you just bought new primers, check for their reliability
(bad primer synthesis ?)
Increase primer amount
Increase template amount
Decrease annealing temperature by 6-10º C and check if
you get any product. If you don't, check all your PCR
ingredients. If you do get products (including unspecific
ones) reaction conditions as described above.
Combine some/all of the above
My PCR product is weak. Is there a way to increase the yield?
Gradually decrease the annealing temperature to the lowest possible.
Increase the amount of PCR primer
Increase the amount of DNA template
Increase the amount of Taq polymerase
Change buffer (KCl) concentration (higher if product is
lower than 1000bp or lower if product is higher than 1000bp)
Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final
concentration) DMSO or glycerol.
Check primer sequences for mismatches and/or increase
the primer length by 5 nucleotides
Combine some/all of the above
My two primers have very different melting temperatures
(Tm) but I cannot change their locus. What can I do to improve PCR amplification?
An easy solution is to increase the length of the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3' end. If size is not a concern, add a few bases at either the 3' or the 5' end of that primer.
I have a number of primer pairs I would like to use together. Can I run a multiplex PCR with them?. How?
Very likely, yes.
Try amplify all loci seaprately using the same PCR program. If one of the primer pairs yields unspecific products, keep the cycling conditions constant and change other parameters as mentioned above (#1 and #2).
Mix equimolar amounts of primers and run the multiplex reaction either in the same cycling conditions or by decreasing only the annealing temperature by 4º C.
If some of the loci are weak or not amplified, troubleshoot.
How many loci can I amplify in multiplex PCR at the same time?
Difficult to say. The author has routinely amplified from 2 to 14 loci. Literature describes up to 25 loci or so.
One or a few loci in my multiplex reaction are very weak or invisible. How can amplify them?
The first choice should be increasing the amount of primer for the "weak" loci at the same time with decreasing the amount of primer for all loci that can be amplified. The balance between these amounts is more important than the absolute values used !!.
Check primer sequences for primer-primer interactions
Short PCR products in my multiplex reaction are weak. How can I improve their yield?
Increase KCl (buffer) concentration to 1.2x-2x, but keep
MgCl2 concentration at 1.5-2mM
Decrease denaturing time
Decrease annealing time and temperature
Decrease extension time and temperature
Increase amount of primers for the "weak" loci while
decreasing the amount for the "strong" loci.
Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final
concentration). You can also try 5% (v/v, final
concentration) DMSO or glycerol
Combine some/all of the above
Longer PCR products in my multiplex reaction are weak. How can I improve their yield?
Decrease KCl (buffer) concentration to 0.7-0.8x, but keep
MgCl2 concentration at 1.5-2mM
Increase MgCl2 concentration up to 3-4.5 mM but keep
dNTP concentration constant.
Increase denaturing time
Increase annealing time
Decrease annealing temperature
Increase extension time and temperature
Increase amount of primers for the "weak" loci while
decreasing the amount for the "strong" loci
Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final
concentration). You can also try 5% (v/v, final
concentration) DMSO or glycerol
Combine some/all of the above [Show Less]