Biology 212/213 Laboratory: Protein Quantitation

Biology 231 Laboratory: Investigations of Human DNA Polymorphisms I: DNA Extraction and Amplification

Background:

Polymorphic DNA refers to chromosomal regions that vary widely from individual to individual. By examining several of these regions obtained from an individual, one may determine a DNA fingerprint for that individual. DNA fingerprints are now widely used to determine paternity/maternity, kinship, identity of human remains, criminal offenders, and the genetic basis of various diseases. DNA fingerprinting was developed by Dr. Alex Jeffreys in 1984 in the United Kingdom and was used in the first murder case in 1987.

The polymerase chain reaction is now used to amplify relatively small amounts of polymorphic DNA from human tissue samples prior to analysis. PCR was invented in 1984 by Kary Mullis at the Cetus Corporation in California. Dr. Mulllis was awarded the Nobel Prize for his work in 1993. The PCR process uses an enzyme known as Taq polymerase to copy a DNA template. The enzyme is purified from bacteria originally isolated from hot springs and so is very stable at high temperatures. Also included in the PCR reaction mixture are 2 synthetic oligonucleotides (15-30 nucleotides) known as primers and the extracted DNA known as the template. The primers are complementary to DNA sequences adjacent to the region to be amplified, known as the target. Finally, the reaction mixture includes free nucleoside triphosphates monomers to be incorporated into the new complementary DNA strands

In the first step of the PCR reaction, the template DNA strands are separated (denatured) from each other at 94oC. In the second step, known as annealing, the sample is cooled to an intermediate temperature (usually 40-65 oC) ANNEALING IS WHATEVER THE MANUFACTURER SAYS to allow hybridization of the primers to the 2 strands. In the third step, known as extension, the temperature is raised to 72 oC THE TAQ POLYMERASE ACTIVATES AT 72 DEGREES and the Taq polymerase adds nucleotides to the ends of the primers complimentary to the target sequence. (Fig. 1)Fig. 1

The three stepsdenaturation, annealing, and extensionconstitute one PCR cycle. This process is usually repeated for 30-40 cycles, amplifying the target sequence exponentially. PCR is performed in a thermal cycler, an instrument that is programmed to rapidly heat, cool, and maintain samples at designated temperatures for varying amounts of time. (Fig. 2)Fig. 2

In todays laboratory, you will extract DNA from your hair follicle cells and set-up PCR reactions for a target DNA sequence from this sample. The target is the PV92 locus on chromosome 16. This locus may or may not have a human-specific transposable element called the Alu insertion(300 BASE PAIRS LONG). Since your body cells contain 2 sets of homologous chromosomes (one from your mothers egg and one from your fathers sperm), you will have two PV92 regions, one on each homologous chromosome. A person may have Alu insertions on both homologous chromosomes or the absence of the insertion on both chromosomes (homozygous) or a person may have an Alu insertion on one homolog but not on the other (heterozygous). Alu insertions are classified as SINES, Short INterpersed elements that are retrotransposons. Human chromosomes contain about 1 million Alu copies, comprising approximately 10% of the genome. All Alu insertions are about 300 base pairs in length so a PV92 region with an Alu insertion will be larger than one without the Alu insertion.

Today, you will also prepare a 1.5% agarose solution to use for making gels in the next laboratory. The gel will be used to separate your PCR products according to size. Agarose is a natural polymer extracted from seaweed. Agarose gels have large pore size and are used primarily to separate large molecules such as DNA with molecular masses greater than 2000 kdal (one kdal = 1000 dal and one H atom = 1 dal). When agarose is heated to about 90 oC it melts, but solidifies again when cooled below 45 oC. During the solidification process, a matrix forms with microscopic pores. The size of the pores vary with the agarose concentration used, typically .05%2.0%. The lower the concentration, the larger the pore size and the larger the DNA fragments that can be separated. Your PCR products should range from approximately 700 base pairs (bp) to 400 bp which will be adequately separated with a 1.5% agarose gel. SMALLER CONCENTRATION, LARGER PORE SIZE. LARGER PORE SIZE, SMALLER AGAROS.

Procedure:

1. Isolate DNA from Hair Follicles:A. Collect 10-20 hair strands plucked from your eyebrows or head containing a sheath, a barrel-shaped structure (often white in color) encircling the shaft near the base of the hair for each PCR reaction. Look at the strands with the dissecting microscope to verify that at least 5 strands contain sheaths. The presence of sheaths is crucial to the success of the PCR amplification since the sheaths contain follicle cells from which the template DNA will be extracted. B. If necessary, cut the shaft end of the strands so that sheaths can be easily placed in the bottom of the tube. Be careful when cutting, the end with the sheath may jump away when cutting the shaft. Place the strands into a 1.7 ml flip top centrifuge tube with the sheaths pointed downward. Use a toothpick to push the strand to the bottom of the tube.C. Add 100 l extraction buffer to the tube containing hair strands. Extraction buffer contains 10 mM Tris-HCL, which maintains pH at 8.3 and increases ion concentration to disrupt noncovalent interactions, detergents, which disrupt cell membranes, and 60 g/ml proteinase K, which will digest proteins and thus release the DNA from the histone proteins. D. Make sure the hair sheaths are completely submerged in the solution and are not stuck to the sides of the tube. Reposition the strands if necessary using forceps or a toothpick.F. Mark your initials on the tube and place in a 55oC waterbath for 1 hr. While the tubes are incubating, proceed to STEP #3 Prepare the 1.5% agarose solution.G. Remove the tube from the waterbath and allow to cool for 30 sec.H. Flick the tube with your finger for 15 sec to mechanically dislodge the cells.I. Check again that the sheaths are completely submerged in solution and that the strands are not stuck to the side of the tube. Reposition if necessary. J. Place the tubes in a float and place the float in boiling water for 10 min. Boiling is necessary denature the proteinase K and inactive it.K. Remove the tube from the boiling water and cool on ice for 2 min.L. Flick the tube with your finger for 10 sec.M. Place the tube in a microcentrifuge. Space the tube evenly apart. Spin for 30 sec. N. Mark an empty 0.2 ml PCR tube with your initials.O. Carefully remove 5 l of supernatant containing your DNA from the 1.7 ml tube and place into the 0.2 ml PCR tube.N. Place the PCR tube containing the 5 l supernatant on ice.

2. Set-up the PCR reactions:A. Add 20 l PCR reaction mix to the PCR tube containing 5 l DNA : Each 20 l of reaction mix contains:EconoTaq 2X Master mix (Taq, dNTPs, GREEN tracking dyes) = 12.5 l- WILL BE IN A MASTER MIXe Nuclease free water----------------------------------------------------------------------7.0l forward and reverse primers = 0.25 l eachB. Gently flick the PCR reaction mixtures and quick spin in the microcentrifuge to collect the entire sample at the bottoms of the tube. Use empty tubes as holders for the PCR tubes.C. Place your tubes into the thermal cycler. The program will run as follows: Initial denaturation: 94oC for 5 min32 cycles: 94oC for 30 sec (denaturation), 61oC for 30 sec (annealing), 72oC for 45 sec (extension)Final extension: 72oC for 4 min D. After the cycling is complete, an instructor will freeze the tubes until next laboratory.

3. Prepare the 1.5 % agarose solution: A. Each group will need 25 ml of 1.5% agarose in Tris-acetate EDTA, pH 7.8 buffer. Since we have 6 groups we will need to make a total of 150 ml. Add 2.25 g agarose powder to 150ml TAE buffer in a heat proof container.B. Swirl the mixture to disperse agarose clumps. Mark the level of the solution on the outside of the container.C. Heat the agarose solution in a microwave oven 1 min at a time until the agarose has completely dissolved. The solution is ready when it appears clear like water. Check the solution carefully. If you see any crystals, continue heating.D. Cool the solution to 55oC with swirling to evenly dissipate the heat. If evaporation has occurred, add de-ionized water to bring the solution up to the original volume marked. Cover the container with plastic wrap, add a piece of tape with your section # and place in frig until next lab.

Modified from EDVOTEK, The Biotechnology Education Company