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Unformatted text preview: highly dependent on temperature. In this lab exercise, a 60°C
annealing temperature is optimum for primer binding.
During the extension step, the job of Taq DNA polymerase is to add nucleotides (A, T, G, and
C) one at a time to the primer to create a complimentary copy of the DNA template. During
polymerization the reaction temperature is 72°C, the temperature that produces optimal Taq
polymerase activity. The three steps of denaturation, annealing, and extension form one “cycle”
of PCR. A complete PCR amplification undergoes 40 cycles.
The entire 40 cycle reaction is carried out in a test tube that has been placed into a thermal
cycler. The thermal cycler contains an aluminum block that holds the samples and can be rapidly
1 Bio 05LA – Fall Quarter 2012 Lab 8 heated and cooled across broad temperature differences. The rapid heating and cooling of this
thermal block is known as temperature cycling or thermal cycling. 3' 5' 5' 3' Denature strands at 94°C
5' 3' 3' 5' Anneal primers at 60°C
(Taq polymerase recognizes 3' ends
of primers) 3' 5' Primer
3' 5' Taq polymerase 3' 5' Primer
5' 3' Extend at 72°C
(Synthesize new strand) 5' 3'
3' 5' 5' 3'
5' 3' Repeat cycle 40 times
Fig. 1. A complete cycle of PCR. The Target Sequence
The human genome contains small, repetitive DNA elements or sequences that have
become randomly inserted into it over millions of years. One such repetitive element is called the
“Alu sequence”. T hi s i s a D N A s equenc e a bou t 3 0 0 b as e pairs lon g t ha t i s repeated almost
500,000 times throughout the human genome. The origin and function of these repeated
sequences is not yet known.
In this laboratory activity you will look at an Alu element in the PV92 region of
chromosome 16. This particular Alu element is dimorphic, meaning that the element is present in
some individuals and not others. Some people have the insert in one copy of chromosome 16 (one
allele), others may have the insert in both copies of chromosome 16 (two alleles), while some may not
have the insert on either copy of the chromosome (Figure 2). The primers in this kit are designed to
bracket a sequence within the PV92 region that is 641 base pairs long if the intron does not contain
the Alu insertion or 941 base pairs long if Alu is present. This increase in size is due to the 300 base
pair sequence contributed by the Alu insert. 2 Bio 05LA – Fall Quarter 2012 Lab 8 When your PCR products are electrophoresed on an agarose gel, three distinct outcomes
are possible. If both chromosomes contain Alu inserts, each amplified PCR product will be 941 base
pairs long. On a gel they will migrate at the same speed so there will be one band that
corresponds to 941 base pairs. If neither chromosome contains the insert, each amplified PCR
product will be 641 base pairs and they will migrate as one band that corresponds to 641 base
pairs. If there is an Alu insert on one chromosome but not the other, there will be one PCR product
of 641 base pairs and one of 941 base pairs. The gel will reveal two bands for such a sample. Fig. 2. The presence or absence of the Alu insert within the PV92 region of chromosome 16. PV92 Genotype DNA Size of PCR Products ALU Homozygous (+/+) 941 base pairs Homozygous (–/–) ALU 641 base pairs ALU Heterozygous (+/–) 1 2 3 4 5 941 and 641 base pairs 6 7 8 (bp)
100 Fig. 3. Electrophoretic separation of DNA bands based on size. EZ Load DNA molecular mass ruler, which
contains 1,000 bp, 700 bp, 500 bp, 200 bp, and 100 bp fragments (lane 1); two homozygous (+/+) individuals with
941 bp fragments (lanes 2, 6); three homozygous (–/–) individuals with 641 bp fragments (lanes 3, 5, and 8), and
two heterozygous (+/–) individuals with 941 and 641 bp fragments (lanes 4 and 7). 3 Bio 05LA – Fall Quarter 2012 Lab 8 Electrophoresis separates DNA fragments according to their relative sizes. DNA fragments are
loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A
direct current is passed between wire electrodes at each end of the chamber. DNA fragments are
negatively charged, and when placed in an electric field will be drawn toward the positive pole and repelled
by the negative pole. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA
fragments can move more easily than larger ones. Over a period of time, smaller fragments will travel farther
than larger ones. Fragments of the same size stay together and migrate in what appears as a single “band”
of DNA in the gel. In the sample gel above (Figure 3), PCR-amplified bands of 941 bp and 641 bp are
separated based on their sizes. Lab Period 2 – Separation and Visualization of PCR Products
The PCR products you generated last week will be separated by gel electrophoresis. After
for loading samples into the wells of an agarose gel (A). The lid is then placed on the apparatus and the
power cords are plugged into the power supply (B). Separation of the fragments will occur as the
current flows through the gel. Note that the cords are color coordinated. The red cord represents the
positive end of the field and the DNA will migrate toward that end of the gel (the dye front will be your
visual indicator). After about 40 minutes at 120V the electrophoresis will be stopped and the gel
transferred to a tray containing a concentrated staining solution (C). The DNA in the gel will bind the
dye allowing visualization of the bands. A C B Can you determine whether you are homozygous (+/+), homozygous (-/-) or heterozygous?
Genotype determined ____________________________________ *The introductory information and protocol has been adapted from “Biotechnology Explorer™- Chromosome 16: PV92 PCR Informatics Kit Manual which is a product of Bio-Rad. Duplication of any part of
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.
- Fall '12