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Primers – short sequences of DNA that are complementary to DNA one wishes to amplify. There are 2 types: forward and reverse. They are separated by the base pairs in the sequence of DNA being amplified. The size of the PCR product is equal to the size of the amplified region of DNA plus the number of base pairs in the primers.
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Buffers and cofactors needed to make the reaction take place at an optimal rate.
4. Why do you need to perform PCR on DNA evidence from a crime scene?
PCR is needed because there is usually not enough DNA obtained from a crime scene to analyze or visualize. 5. What steps make up a PCR cycle, and what happens at each step? Each PCR cycle is made up of 3 steps.
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Denaturation – the DNA strands are melted apart.
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Annealing – primers bind to complementary sequences on the DNA.
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Extension – DNA polymerase adds nucleotides to primers.
Lesson Two: Electrophoresis of PCR products 1. Why does DNA move through an agarose gel?
Since DNA is negatively charged, it can be separated using an electric current. In fact, electrophoresis means "carry with current". Movement through the gel occurs when an electric current is applied across the gel. Since the gel is immersed in buffer, the current will travel through the buffer and gel, carrying the negatively charged DNA with it toward the positive anode. 2. What are the two techniques used to create a DNA profile? What function does each perform?
PCR and gel electrophoresis are used to create a DNA profile. PCR is used to amplify sufficient amounts of a DNA sample to be analyzed. Gel electrophoresis separates bands based on size. After the bands are separated the gel is stained to visualize the band pattern. By comparing the bands in the gel to a standard, we can estimate their size. 3. What is an allele ladder? What is its function in DNA profiling?
An allele ladder is a mixture of the alleles possible at a particular locus. The Allele Ladder is needed to identify the PCR products (alleles) present in the evidence obtained from the crime scene. 4. What is required to visualize DNA following electrophoresis?
DNA is visualized by applying a stain to the gel. In this exercise, Fast Blast DNA stain is used, which turns DNA present in the gel an intense blue color.
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