BIO 150
Spring 2009
LAB PRACTICAL 2
STUDY GUIDE
Questions:
1.
Item to view: An electropherogram showing the results from the genotyping of 4 STR
loci: D8S1179, D21S11, D7S280, and CSF1PO.
Question: Using the Caucasian allele frequency table provided, calculate the
combined Pm for this partial Identifiler profile. Show all of your calculations.
Locus
D8S1179
Genotype
15,16
D21S11
30,30.2
D7S820
9,11
CSF1PO
11,11
Calculation
Pm
Combined Pm
*2.
Item to view: Nothing (refer to answer in Question 1).
The profile in question #1 is that of an alleged father in a paternity test. The profiles
of the mother and child at these same loci are shown in the table below.
Locus
D8S1179
D21S11
D7S820
CSF1PO
Mother
12,17
28,29
10,10
11,15
Child
12,15
28,29
9,10
11,11
Questions:
3.
a.
Underline each of the obligate paternal alleles in the child’s profile. If an OPA
cannot be determined, underline both alleles carried by the child.
b.
Is this man included among possible fathers for the child? If so, calculate the
Combined Paternity Index generated by this paternity test. Show all your
calculations. If not, explain why.
Item to view: The blunt end of a rooted hair under 10X on a light microscope.
Question: Look through the microscope. What are you looking at? Be as specific as
possible.
Page 1
BIO 150
4.
Spring 2009
Item to view: CODISmt printout.
Questions:
Examine the CODISmt printout and then answer the following questions:
5.
a.
Define the term SNP. How do SNPs relate to mtDNA haplotypes?
b.
Calculate the frequency of this mtDNA haplotype in peoples of African origin.
Show your calculation and express your final answer as a FRACTION.
Item to view: An electropherogram with high peak height and split peak artifacts.
Questions:
Examine the electropherogram.
*6.
a.
What type of artifact do you see in this profile?
b.
What failed to occur during PCR that caused this to happen? Be as specific as
possible.
c.
What would be the best way to fix the problem if you were to re-amplify the
sample?
Item to view: Nothing
Question: Explain how ethidium bromide works as a stain for visualizing DNA.
*7.
Item to view: Nothing
Questions:
5' - A T G T A A G A T G G C - 3'
8.
a.
Calculate the melting temperature of this PCR primer. Show your calculation.
b.
What size would a genome need to be in order for this primer to be specific
enough for use in a PCR reaction? Show your calculation.
Item to view: Micropipettor set for 6 uL
Questions:
9.
a.
For what volume is this micropipettor set? Express your answer in uL, mL,
and L.
b.
What is the highest volume that you can accurately pipette using this
micropipettor?
Item to view: A printout of the results of a q PCR run.
Page 2
BIO 150
Spring 2009
Questions:
Examine the qPCR printout.
10.
a.
What is the concentration of human DNA in the sample indicated by the arrow?
Be sure to include units!
b.
Assuming that this sample was diluted 1:75 prior to quantification, calculate
the volume of undiluted DNA extract you would need to add to a Minifiler
reaction to ensure that the amplified product would equal exactly 0.1 ng. Can
you micropipette this volume accurately? If not, what could you do to remedy
the situation? (NOTE: Minifiler reactions can only accommodate up to 10 uL of
DNA.)
Item to view: Photo with a gel containing two bands (A and B).
Questions:
*11.
a.
What are the two functions of the loading dye used during agarose gel
electrophoresis?
b.
Examine the photo of the gel. Which band of DNA has a higher molecular
weight? Explain.
Item to view: Nothing.
Question: The mutation rate of mtDNA is 10 times higher than that of nuclear DNA.
What are the two primary reasons for this?
*12.
Item to view: Nothing.
Question: After generating an STR profile from a genotype, an analyst must always
check over the data and may make manual adjustments. Fill in the table below,
listing 3 biological and 3 technological reasons why manual intervention is necessary.
Biological
*13.
Technological
Item to View: Nothing.
Questions:
A single, rooted hair is found in a black ski cap left at the scene of a robbery. STR
typing of the hair yields a full profile that matches the profile generated from a
buccal swab taken from the main suspect in the case - with the exception of the
alleles at one locus, D7S820. At this locus, the hair types as (10,12) while the buccal
Page 3
BIO 150
Spring 2009
swab DNA types as (10,12,13), with the 13 allele being only about ¼ the peak
height of the other two.
*14.
a.
Assuming that the 13 allele is reproducible when the buccal swab DNA is
analyzed a second time, what is the most likely explanation for these results?
b.
As an analyst, what could you do to test whether your explanation is correct?
Item to View: Nothing.
Questions:
Below is a human DNA sequence and you wish to amplify the entire sequence using
PCR.
5'-AAGAGATAGATCTCTCCCCTCGCTGCTCGTACTCGCTCGATCTACCCAACTCTAGCTCATTCCTC-3'
3'-TTCTCTATCTAGAGAGGGGAGCGACGAGCATGAGCGAGCTAGATGGGTTGAGATCGAGTAAGGAG-5'
*15.
a.
Design a set of primers to accomplish this task. Label the 5' and 3' ends of
each primer.
b.
What annealing temperature would you use in your PCR reaction? Briefly
explain how you arrived at your answer.
Item to View: Nothing.
Question: During the QIAGEN QIAamp DNA spin column DNA extraction procedure,
chemical waste was generated. What was present in this waste that made it toxic?
*16.
Item to View: Nothing.
Question: In the space below, write out the sequence of the 11.3 allele of any typical
human DNA identification STR marker.
EQUATIONS:
Pm = p2 + [(p)(1-p)(0.01)
Pm = 2pq
Ppopulation = Pdatabase + 1.96
1/2
(Pdatabase)(1- Pdatabase)
N
Pdatabase = X/N
Ppopulation = 1 - 0.051/N
PI = 0.5/(freq OPA)
PI = 1/(freq OPA)
Page 4