A501- Fall 2006
Assessing relatedness- Genotyping with microsatellites
Thursday Sept. 14: Introduction to assessing relatedness
Please read:
Alberts et al. 2002. Molecular Biology of the Cell, Chapter 8, “Manipulating Proteins, DNA, and
RNA”; focus on first section, and section discussing PCR,
(http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.section.1553 ); Table 8.7,
(http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.table.1554); Figure 8-39,
(http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1590 ).
Then view PCR movie at: http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html
Selkoe, K.A. and R.J. Toonen. 2006. Microsatellites for ecologists: a practical guide to using and
evaluating microsatellite markers. Ecol. Letters 9(5): 615-629.
Tuesday Sept. 19: PCR and genotyping of DNA sample
The concentration of your bird DNA has been determined using a microplate spectrophotometer (see:
http://cgb.indiana.edu/genomics/equipment/3 for more information). Using the concentration given to
you, bring your tube to a standard concentration of 100ng/ul, in sterile, distilled water.
For example:
If your DNA concentration is 890ng/ul, and you want a final concentration of 100ng/ul
in a volume of 200ul, then what volume of the undiluted DNA solution would you add?
Hint: 100ng/ul = (890ng/ul)(x ul)
200ul
Once “x” is known, subtract this value from 200 to obtain the amount of water to add.
Note- if your original concentration is less than or equal to 100ng/ul, you do not need to dilute it.
After you have standardized your DNA, you can use this working solution in a PCR reaction to
amplify a microsatellite marker, or a gene locus. For instance, if you wanted to study factors affecting
the rate of extra-pair copulations in juncos you could use microsatellite data to assign parentage to
individuals within a clutch.
We will amplify the Dpu16 marker, which was originally isolated from the yellow warbler
(Dendroica petechia), but is known to work on juncos.
1. In order to run the PCR reaction, you must first mix the reagents in an Eppendorf tube, and then
aliquot a set amount into a thin-walled PCR tube. Mix the reagents in the following order, after
calculating the amount of each you will need, based on the number of samples in the class:
Water
10X PCR buffer
Magnesium Chloride (MgCl2) (25mM)
Deoxynucleotides (dNTP’s) (2mM)
Forward primer (20uM)
Reverse primer (20uM)
Taq polymerase (keep on ice)
(DNA-do not add in this step)
Total reaction volume
1X
5.70ul
1.0
1.0
1.5
0.1
0.1
0.1
(0.5)
10.0ul
?X
2. Take 9.5ul from the Eppendorf tube, and dispense into a PCR tube. Repeat for each sample, plus one
more for the negative control (blank).
3. Add 0.5ul of your standardized DNA to your PCR tube, and place tube in PCR machine. In
addition, add 0.5ul of water to the blank tube.
4. Place your sample tubes in the PCR machine. It will then run the reaction for this marker, which
consists of the following steps:
1. 94oC x 3.00min
2. 94oC x 30 s
3. 50oC x 30 s
4. 72oC x 30 s
5. Repeat steps 2-4, for 29 cycles
6. 72oC x 5.00 min.
5. When complete, each reaction will be diluted and mixed with a DNA ladder (ladder contains DNA
fragments of known sizes, increasing from small to large pieces, which is useful for sizing an
amplified gene or marker fragment; see: http://www.promega.com/figures/popup.asp?fn=1836tc
for an example).
6. This mixture will then be run on an automated, capillary sequencing machine, known as an
ABI 3730, which will separate the amplified microsatellite alleles by size. (See
https://products.appliedbiosystems.com/ab/en/US/adirect/ ab?cmd=catNavigate2&catID=600533
for an image/description).
Thursday Sept. 21: Data analysis/ (Introduction to measuring plasma hormone levels, EIA/RIA)
1. Today you will download your microsatellite data from Tuesday, and analyze the results using a
program called GeneMapper.
2. Refer to the Genemapper instructions handout as you load and analyze your data.