pv92 pcr finding your alleles. listed here are functional genes (genes that code for proteins). pv92...
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PV92 PCR
Finding Your
Alleles
Listed here are functional genes (genes that code for proteins).
PV92 is not functional (as far as we know) so it is not shown. It is an intron.
PV92 is 601 base-pairs long. Some PV92 segments carry ONE Alu SINE (Single INterspersed Element; aka: Alu repeats)Each Alu is about 300 base-pairs long. So PV92 with an Alu would be ______ base-pairs long.
10% of your genome is made of Alu SINE!
Possible genotypes:
+ -
Alu repeat
Alu repeat
Alu repeat + +
- -
Locus = location of a gene or element
Finding your genotype
1. Get DNA out of hair cells
2. PCR the PV92 segment.
3. Gel Electrophoresis of amplified DNA regions with (or without) the Alu repeat.
Need a “hair tag”
Get DNA out of Hair Cells• Instagene Matrix has beads that cling to Mg2+ .
Without the Mg2+ available, the DNAases that will come out of lysosomes won’t work (which is good, because then the DNAases can’t degrade your DNA!)
Mg2+
Mg2+
DNAases
DNA
Normal Conditions: Virus DNA enters cell, DNAases are released from lysosome, Mg2+ activates the DNAases, & goodbye virus DNA!
nucleus lysosome
Get DNA our of Hair Cells
• Heating unclumps the cells and helps deactivate the DNAases.
• High heat breaks down all membranes (cell, nucleus, lysosomes, etc.) so DNA is released.
PCR = Polymerase Chain Reaction• MASTER MIX HAS
• Primers – Forward and reverse, that will latch on to the top strand and the bottom strand of DNA at 60O (after it is denatured at 92O).
Forward Primer
Reverse Primer
Master Mix has
• Taq polymerase – at 72O this enzyme will add As, Ts, Cs, and Gs to the DNA where the primers leave off.
• Nucleotides (As, Ts, Cs, and Gs) for Taq polymerase to add.
• MgCl2 – to stabilize DNA.
Taq polymerase
Taq stands for Thermus aquaticus, a bacterium that lives in hotspring. To do PCR, DNA must be split apart (denatured). This can be done using high heat. So the enzyme that puts nucleotides back on to the DNA has to be able to withstand high heat. Enzymes in bacteria that live in hotsprings can do this.
PCR Annimation
• http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
Denature – 92O
Anneal – 60O
Extend (or Polymerize) – 72O
Gel Electrophoresis
• Agarose acts as a “molecular sieve.” Small things pass through it easily, larger things don’t.
• The smaller the DNA fragment, the faster it runs.
• DNA has a - charge, so you put the wells with DNA at the – end and the DNA will be attracted to the + end.
Ladder (molecular mass ruler, or MMR)?
Gel ElectrophoresisResults
Loading dyes are added to every sample, including the controls, so that (1) the sample sinks to the bottom of the well (due to the glycerol in the dye) and (2) so you know when your run is complete. Because the loading dye is so small, it will run faster than any of the DNA samples. When the loading dye gets near the end, it’s time to quit the run before you DNA runs off the end!
Molecular Mass Ruler
Gel ElectrophoresisResults
Molecular Mass Ruler (MMR) is placed in the first well. It is made of several pieces of DNA of known length. You can compare your DNA lengths to the MMR to estimate the lengths of your DNA.You can also just compare your DNA lengths to the known + +, + -, and - - . But it is standard procedure to always use a MMR.
Genotype Frequency of Class
Number of Students
Frequency of genotype
(# of students with genotype/total # of students)
+ +
+ -
- -
Total # of students =
Genotype Frequency US population
Number of individuals
Frequency
+ / + 2,422 0.24
+ / - 5,528 0.55
- / - 2,050 0.21
Total 10,000 1.00
How does our class compare?
Total number of Alu alleles in class
Number of students
Total number
of Alu alleles
+ + +
- + -
- -
Total # of alleles =
Frequency ofAlu alleles
+-
Frequency ofAlu alleles
+-
Total number of Alu alleles in US population
How does our class compare?
Number of individuals
Total number of Alu alleles
+ + 2,422 +
- + - 5,528
- - 2,050
Total =
Your Gel Sketch