Lecture 12/3 - Start Final Flashcards
What method is usually used to analyze mtDNA
Cycle sequencing
what are the usual samples
hair, teeth, bones
Is nDNA and mtDNA usually processed in the same facilities?
No, separate bc mtDNA is rlly sensitive to contamination
AFDIL and AFMES (AFDIL came first)
Armed Forces DNA Identification Labratory ->
Armed Forces Medical Examiner System
What did they do (AFDIL/AFMES)
develop pcr and cs protocols and primers etc. for human mtDNA
what is used to grind up hair
tissue homogenizer
How many base pairs is in D-Loop
1000 bp
How many base pairs in mtDNA
16,569
PS, MPS
Primer set, Mini primer set
What are the 3 issues that cause trouble when working with mtDNA
- Interpretation of a perfect match (maternally inherited)
- Heteroplasmy
- Contamination
Why is interpretation of a perfect match an issue when analyzing mtDNA
not unique TO PERSONS, maternally inherited,
power rule does not apply bc haploid locus
Unique haplotypes are common
SWGDAM guidelines
Exclusion (2+ nt differences)
inconclusive (1 nt difference)
cannot exclude (identical sequence/ no diff)
SWG DAM
Scientific working group DNA analysis Methods
What is a hot spot
A location that is too variable, can cause “failure to exclude” shouldn’t be useddddddddddd
Proportion 95% confidence interval (of database for N)
p(hat?) +/- 1.96 (SQRT p(1-p)/N)
What is heteroplasmy? Sequence and length?
The detection of >1 haplotype in a single organism
Sample has two different haplotypes within it, so one point mutation (SEQUENCE) or indel (LENGTH, one more base)
What does the number after F or R tell you?
The base position at the 5’ end
What are you looking for to exclude from your sequence after sequencing the forward primer amplicons
The reverse compliment of the reverse primer (6-7 b)
remember homometric stretch of C’s
associated w length heteroplasmy
What is the threshold of contamination
contamination < 1/10 of the sample DNA aka doesn’t affect the haplotype
DGGE
Denaturing gradient gel electrophoresis
