Lecture 9/14 - E2 Flashcards
Common Temperature for Denaturation
94-98C
Common Temperature for Annealing
37-60C
Common Temperature for Extension
68-72C
How do you calculate the total number of amplified products after each cycle?
2^n
n = the number of cycles
How do you calculate the total number of ds DNA products when you begin with a single ds DNA molecule?
A(2^n - 2n)
A = the number of starting ds DNA template molecules
n = number of cycles
Advantages of PCR over traditional approaches?
Faster and less expensive, can be optimized to detect low abundance mRNA splice variants, and it can be used for other purposes
Limitations of PCR technology
Requires some precise knowledge of target DNA sequence in order to create the primers, size restrictions on amplicons, mutations may be introduced into the DNA by the polymerase during amplification
Starting with genomic dsDNA isolated from a single somatic cell, how many fully dsDNA products are present after 4 cycles of PCR?
A) 0
B) 2
C) 4
D) 6
E) Cannot determine based on the information providded
E
Need to know how many molecules are present at the start of the reaction which depends upon the stage of the cell cycle
Starting with genomic dsDNA from a single somatic cell at the G1 stage of the cell cycle, how many fully dsDNA products are present after 4 cycles of PCR? A) 0 B) 4 C) 8 D) 16 E) 32
There are 2 tmplates because of mom and dad copies
A(2^n - 2n)
2(2^4 - 2(4)) = 2(16-8) = 16
Haplosufficient
Where one copy of the WT allele is needed for the full WT functionality (Aa)
Haploinsufficient
Where one copy of the fully functioning allele shows a mutant phenotype (Aa = mutant)
Null/Amorphic Mutations
Complete loss of function mutation that produces no functional gene product
Completely inactivate gene function
Weak/Leaky/Hypomorphic Mutations
Mutations that result in partial loss / reduced function
- Depends on the level of activity of the leaky mutant allele
Possible defects associated with Null Alleles
Gene absent or inactivated No transcription Abberrant proessing of the mRNA transcript No translation Non-functional protein product
Possible defects associated with Hypomorphic Alleles
No gene Reduced rate of transcription Abberant processing of the mRNA transcript Reduced translation Less-functional protein product
Which types of DNA change in the yeast ACT1 gene is most likely to result in a hypomorphic mutation?
A) Deletion of all ACT1 protein coding sequence
B) Removal of all teanscriptional regulatory sequences
C) A codon change resulting in an amino acid substitution
D) ACT1 gene duplication
E) All equally likely to cause hypomorphic mutation
C) A codon change resulting in an amino acid substitution
Because it is the least mild change
Hypermorphic Allele
Increased activity (more is not always better)
Neomorphic Mutation
Mutation that results in an increased function
Dominant Negative Mutation
Competes with the WT protein and blocks natural function
Produces poisonous gene products
“Spoiler Effect” - Homo dominant can be lethal and too toxic
Which type of mutation is most likely to result in a haploinsuficient genotype? A) A null allele B) A weak hypomorphic allele C) A wildtype allele D) A gene duplication
A) A null allele
