Lecture 9 Flashcards
What are the four types of artifacts?
- Dye blob
- Pull up
- Stutter
- Spike: All channels
What is an artifact
Not a real allele
What are the common issues with PCR CE that are NOT artifacts?
- Minus A, Split peak, incomplete adenylation
- Off ladder
- Allele dropout
Stutter
A repeat that is one or more repeat less than the true allele, less than 15% of the true allele
Unavoidable
Bc of PCR slippage = one repeat loop
varies across loci and allele
Longer alleles have more stutter bc more t in extension = more opportunity for stutter
What type of STR has too much stutter to be used in forensics?
Dinucleotide
(stutter is a function of repeat length)
Off ladder is another word for
Microvariants
What base does PCR polymerase tack on the end of ea amplicon?
Adenine
What is minus A, split peak, or incomplete adenylation?
When there is not enough time during extension or if there’s too much template DNA
Might be mistaken for microvariant
One base pair difference
What are Null/ Silent alleles or allelic dropout
In what samples is it common in?
- present in DNA sample but fails to be amplified due to a nucleotide a change in the primer binding site.
- Can depend on population
- Heterozygous appears Homozygous
- Common in low copy number/ template DNA OR degraded DNA (you can see this where the longer fragments have shorter peaks - “Decay curve”)
What can cause pull up
Excess injection, too much DNA
What could cause variation in mobility across injection (4)
- Buffer not changed often enough
- Dirty intrument shorting out
- Temp change
- Capillary wall becomes charged (changing electro-osmotic force)
How can you combat the small ions that compete with DNA during injection?
Wash with formamide
What does the small ions do
increase time, throws off sizing function = peak shift
