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Lecture 20

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Biology 1202B
Brenda Murphy

1 Lecture 20 Question & Answer Some Information  Biggest issue with PCR is contamination as it only takes one cell to skew the results  Control is very important for any experiment  Positive control – picking something that you know will give you the results you expect  Negative control – expected to give you no results (ie. In PCR no amplification)  Ladder, positive control and negative control are all important  If there is a band in the negative control, check if the PCR worked o If it amplified then it did not work Case Study  A recent paper reports that the presence of an autosomal recessive SNP (guanine changed to adenine) in the “hair texture” gene correlates with curly hair  Looking at the sequence surrounding the genetic variation, you notice that this guanine nucleotide is in an EcoRI restriction enzyme sequence recognition site  In particular, if the guanine nucleotide is changed to an adenine nucleotide, this eliminates a restriction enzyme cut site  You decide to investigate these findings for yourself by screening 6 people (2 people with straight hair & 4 people with curly hair)  You PCR amplify DNA to produce a 300bp fragment which contains the genetic variation in question  If the PCR amplified sequence has a guanine nucleotide at the SNP location, the PCR fragment will cut with EcoRI to produce 100bp and 200bp fragments Straight Hair Curly Hair -- G – -- A -- -- C -- -- T – (maternal or paternal) Eco RI cuts Eco RI Does not 300 |--------------------------| Will produce - |------|--------------| 100 200  Run a gel to look at the bands  Primers flank the area of interest, surround the area of interest o One area will be upstream and another will be downstream  Upstream – towards the 5’ end  Downstream – toward the 3’ end  Genes are not always transcribed off one strand of DNA, (there may be transcription on next strand) – important to determine which strand you are on 2  Many things that you can do to not amplify something  Ultimately, the persons hair may be wavy or may be straight but will not be curly Where are your PCR Primers? 1. Upstream of the SNP 2. Downstream of the SNP 3. Surrounding the SNP 4. Within the SNP Why are you Testing 2 people with straight hair? 1. Negative Control for EcoRI Digestion 2. Positive Control for EcoRI Digestion 3. To increase the number of subjects for statistical purposes 4. Equal number of subjects to experimental If this PCR is successful, what do you e
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