Week 1: DNA Flashcards
Which synthetic probe will be more stable?
Synthetic probe 1 because of the T mismatch in probe 2
How do we test for a mismatch in our probes?
a mismatch decreases the melting temperature so we would increase it and see which one denatures first
A genetic variant in the beta hemoglobin gene leads to Sickle Cell Anemia, (HBB-E6V), in which the 5th codon of the beta-globin encodes valine (Val, or V) instead of glutamic acid (Glu or E). Based on this information which probe will have the higher melting point?
Synthetic probe 2 because it matches the variant mutation
How would someone with Sickle Cell Anemia (SCA) [Glu to Val], a homozygous mutation, display hybridization compared to someone with Sickle Cell Trait, a heterozygous mutation?
a person with Sickle Cell Anemia (SCA) would only show stable hybridization with probe 2 compared to a person with Sickle Cell Trait showing stable hybridization with both probes
What are the 3 basic steps of qPCR?
Denature, Anneal, and Elongate
Shown below is the Annealing step of PCR. Where on this probe would Elongation start?
What sequence of base pairs would DNA polymerase add to this structure?
i.e., 5’-TCAGAGACTTCTC-3’
What is the portion colored in green referred to as?
olignonucleotide
What end is DNA added on to a primer?
3’ end; that way it is sythesized 5’ to 3’
List the sequences of the two primers that would amplify this target
which primers would be useful in identifying whether someone is affected, a carrier, or not affected?
primers 1 and 4 because they include the entire alpha-1 ORF (open reading frame).
What is an ORF (open reading frame)?
The sequence of codons beginning with a start codon, then a series of codons for amino acids, and ends with a stop codon. This sequence can potentially be translated into a polypeptide product that is the entirety of a gene product.
How large would the PCR products of the gene shown need to be to determine whether someone is affected, a carrier, or not affected?
affected - 2 PCR products ~300 bp
carrier - 1 PCR product ~300 bp and 1 PCR product ~1200 bp
not affected - 2 PCR products ~1200 bp
If a patient sample contains 1000 copies of a target molecule being amplified by PCR/NAAT, how many cycles of amplification will it take to get 1 million copies?
10;
Memorize 2^10 = 1024 or ~1000
Exponent math refresher -
1000*1000=1,000,000; so 1,000,000 = 2^10 * 2^10 = 2^20 (add exponents if base is the same)
2^10 + 2^10 = 2^11 (if bases and exponents are the same you add 1 to the exponent)
A patient diagnosed with Chronic Myeloid Leukemia resulting from a translocation involving chromosomes 9 and 22 (the Philadelphia chromosomal translocation, t(9;22)) is being monitored for the effectiveness of the patient’s alternative chemotherapy treatment (for religious reasons they don’t want to take the receptor tyrosine kinase inhibitor imatinib). A quantitative PCR assay using primers that flank the translocation site was performed at diagnosis 6 months ago, with a Crossing point (Cp) of 18.5 (crossing point, this indicates that a specific quantity of PCR product was detected after 18.5 cycles). The same assay performed today gives a Crossing point (Cp) of 17.0. This result is most accurately interpreted to mean that the present level of leukemic cells relative to the level at initial diagnosis has what?
2^1.5= ~3;
less cycles mean more product
Based on the graph below, which sample started out with the most and the least amount of the targe molecule?
Blue - Most
Army Green - Least
Based on the graph, which sample has 1000x less target molecules than the lavendar sigmoid curve?
The teal graph
2^10=1024; if add 10 cycles to the fluorescent threshold 24, you get 34.
What is the sequence for both the top and bottom circled nucleotide?
What do the colors black, red, and green mean in this microarray?
In the “Massively Parallel” or “Next Generation” technique which strand in the diagram represents the sequence, “GGGCGG”
