You receive 12 samples in your laboratory for genetic screening. The samples form two families. The first family is composed of samples: 1(Mom Smith); 2 (Smith Son 1); 3(Smith Daughter 1); 4(Smith Son 2); 5(Smith Son 3); 6(Smith Son 4); 7(Smith Son 5); 8(Smith Dad) and 9(Mom Jones); 10&11(Jones twin boys); 12(Jones Dad). Mom Smith and Dad Jones are siblings.
Please construct the appropriate Pedigree using the guidelines given in class.
Given the following spectrophotometric readings calculate the purity and concentration of the stock solutions.
Desired Ranges:
260/280=1.8-2.0
260/250=1.0-1.2
230/260=0.4-0.5
5uls of stock DNA were diluted with 995 HOH and measured.
| Sample |
A280
|
A260
|
A250
|
A230
|
260/280
|
260/250
|
230/260
|
ug/ul of stock
|
|
1
|
0.189
|
0.350
|
0.313
|
0.151
|
|
|
|
|
|
2
|
0.054
|
0.095
|
0.084
|
0.041
|
|
|
|
|
|
3
|
0.089
|
0.159
|
0.142
|
0.072
|
|
|
|
|
|
4
|
0.111
|
0.201
|
0.180
|
0.092
|
|
|
|
|
|
5
|
0.130
|
0.237
|
0.212
|
0.108
|
|
|
|
|
|
6
|
0.128
|
0.231
|
0.206
|
0.102
|
|
|
|
|
|
7
|
0.032
|
0.048
|
0.043
|
0.029
|
|
|
|
|
|
8
|
0.057
|
0.101
|
0.090
|
0.046
|
|
|
|
|
|
9
|
0.141
|
0.256
|
0.230
|
0.117
|
|
|
|
|
|
10
|
0.122
|
0.220
|
0.196
|
0.098
|
|
|
|
|
|
11
|
0.086
|
0.151
|
0.135
|
0.072
|
|
|
|
|
|
12
|
0.149
|
0.270
|
0.242
|
0.134
|
|
|
|
|
Based on the above information, do you predict any difficulties in performing a restriction digest on any of the above samples? Explain
Based on the information calculated previously set up a digest with each sample that contains 10ugs of DNA/sample and 40 units of Hae III enzyme.
|
Sample
|
uls DNA
|
uls 10XBuffer
|
uls HOH
|
uls Enzyme
|
Total
|
|
1
|
|
|
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2
|
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3
|
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4
|
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|
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5
|
|
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|
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|
6
|
|
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|
|
|
|
7
|
|
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8
|
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9
|
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10
|
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11
|
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|
12
|
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Prepare a 1.25% agarose gel in TAE Buffer to run the above samples. The gel box is 10cm X 25cm. The comb has teeth that are 0.5cm x 0.15cm.
Total amount of TAE:____________________________
Total amount of Agar:___________________________
Review the photo taken to document the electrophoretic gel.
Was it consistent with your predictions:
What could have caused the problems noted:
What could you do to resolve these problems:
Review the following data (presumably from an autoradiograph) and answer the questions provided.
If child 3,4,6 had evidence of disease, how would you interpret the above data?
Can you predict who the carriers are?
Is this consistent with an autosomal dominant, autosomal recessive or sex linked mode of inheritance?
Interpret the following data if this were a paternity test.
Is the data consistent with the information stated previously.
Mom Smith is heterozygous for an X-linked trait that can be recognized by a polymorphism when her DNA is cut with Hae III and probed with ExTend probe. The polymorphism is a 3.5kb band for normal and a 1.5kb band for disease. Please diagram what you predict the autoradiograph would look like for the above samples if the Smith sons 1 and 4 are affected and Smith Dad, Mom Jones and Dad Jones are not affected.
1 2 3 4 5 6 7 8 9 10 11 12
Is the Smith daughter a carrier? Explain.
What if Dad Jones is also affected but not Mom Jones or Dad Smith?
1 2 3 4 5 6 7 8 9 10 11 12