DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes
have the same forward primer and reverse primer
A strip of empty PCR tubes
Disposable pipette tips: only use each only once. Never reuse disposable pipette tips. If you do,
the samples will become cross-contaminated
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G8 +
Positive control
none
G8 -
Negative control
none
G8 1-1
28692, replicate 1
28692
G8 1-2
28692, replicate 2
28692
G8 1-3
28692, replicate 3
28692
G8 2-1
68634, replicate 1
68634
G8 2-2
68634, replicate 2
68634
G8 2-3
68634, replicate 3
68634
DNA Sample Set-up Procedure
Using the materials listed in Lab A, first cut eight empty PCR tubes.
Have one group member collect specific patient samples, the PCR reaction mix, controls, and a tube rack from supply cart.
Then have another group member collect a red plastic cup for discarded tips, a micropipette, a marker for labeling tubes, and scissors from the same cart.
Cut your eight empty PCR tubes into two rows of four PCR tubes.
In order to track the contents of each tube, label the sides of each empty tube with the labels created in Lab A.
Place the empty tubes in a rack so they are secure.
Take the empty tube that will contain the positive control and with a pipette place 50 microliters of the given PCR reaction solution into the tube.
With a new pipette tip, as it is important to discard of the used ones, add primer one to the tube with the positive control DNA until the volume reaches 100 microliters. It is important to be sure the volume is 100 microliters or very close to it.
Repeat steps 4 and 5 for the other reactions samples including the negative control, the patient 1 replicates, and the patient 2 replicates.
Close the reaction tubes after making sure each tube has 100 microliters of each corresponding PCR Reaction.
Take the tubes over the to the thermal cycler and wait to start the machine until all of the slots are full.
OpenPCR program
HEATED LID: 100°C: Hydrogen bonds which hold the DNA strand together are broken to initiate denaturing.
INITIAL STEP: 95°C for 2 minutes: Double-stranded DNA begins to denature and form two single-strands of DNA.
NUMBER OF CYCLES: 25
Denature at 95°C for 30 seconds: Double-strand of DNA is unwound into two single strands of DNA.
Anneal at 57°C for 30 seconds: Primers lock onto a single strand of DNA, which prevent the two single DNA strands from rejoining.
Extend at 72°C for 30 seconds: DNA polymerase is activated to add complementary DNA nucleotides to the original DNA strand.
FINAL STEP: 72°C for 2 minutes: DNA polymerase continues to add DNA nucleotides until it reaches the end of the strand. Once the new double-strand is formed, the DNA polymerase falls off.
FINAL HOLD: 4°C: At 4°C, the DNA strand is frozen to store. This prevents denaturing of the new DNA strand.
Research and Development
PCR - The Underlying Technology
Primers bind to DNA to prevent the now separated DNA strands from rejoining. Once primers are added to the DNA stand, a short DNA strand is added as a starting point for complementary nucleotides to be added.
DNA polymerase slides along the single stand of DNA and adds complementary nucleotides. Once all nucleotides are added and the polymerase reaches the end of the strand, it falls off.
PCR Reaction Components
When using the PCR method to copy DNA molecules, first, a template DNA molecule needs to be obtained. A specific strand DNA molecule is extracted from a subject, and is used as a template for the PCR process. Next, the primers, which are a small section of DNA nucleotides made in a laboratory, bind to sites on the DNA strands on either end of the segment that needs to be copied. After the primers bind to the DNA, a taq polymerase enzyme assembles the nucleotides into strands of DNA. Finally, the dNTP’s supplies the bases ( dATP, dTTP, dCTP and dGTP) to the polymerase enzyme finalizing the process making a complete strand of DNA.
Effects of Thermal Cycling on Components
During the first two minutes, the the DNA is put in 95°C the double helix DNA strand separates into two single-stranded DNA molecules. Then, for 30 seconds at 95°C during DNA denaturation, the double-stranded DNA will separate into two single-strands. Next, for 30 seconds at 57°C the annealing process begins where single-stranded DNA molecules attempt to pair up, but the primers crowd in the tube and lock onto the strand before a double strand can form. During the next process, the DNA extends at 72°C for 30 seconds in which the DNA polymerase is activated, which locates a primer attached to a single-strand of DNA. The DNA polymerase adds complementary nucleotides to the DNA strand until it reaches the end of the strand and falls off. Finally, for two minutes at 72°C the DNA polymerase is stimulated to add complementary nucleotides to the single-strand of DNA by sliding along the single DNA strand. Once completed, the DNA polymerase falls off the newly created double-strand. For later use, the DNA can be stored at 4°C to prevent the it from denaturing.
DNA Base-Pairing
The nucleotide Adenine (A) anneals with Thymine (T) just as Thymine anneals with Adenine, while Cytosine (C) pairs with Guanine (G) and Guanine with Cytosine.
Thermal Cycling
Base-pairing occurs during two steps of cycle one of thermal cycling known as annealing and extending. At the beginning of cycle one, the thermal cycler heats up to 95 degrees Celsius so the DNA double helix can separate and create two single-stranded DNA molecules. The cycler then cools down to 50 degrees Celsius allowing the single-stranded DNA molecules to try and pair up (annealing). However, due to the fact that there are more primer sequences than DNA strands, the primers are able to attach to their targets before the strands reconnect. The cycler then heats back up to 72 degrees Celsius for 30 seconds during the extend period, effectively activating the DNA polymerase so it can add nucleotides onto the single DNA strand until it falls off after pairing the complementary base-pairs.
SNP Information & Primer Design
Background: About the Disease SNP
A single nucleotide polymorphism(SNP) is a variation in the genetic code where a single nucleotide on the same area of chromosome differentiates members of the same species. These mutations have a wide effect on quality of life that spans from life-threatening diseases like sickle-cell anemia to non-threatening disorders like a change in hair color. One specific SNP, found on the Apolipoprotein E (APOE) gene on chromosome 19:44907853, interchanges Cytosine with Thymine and results in Alzheimer's disease. The point mutation that results in this SNP codes for a different codon that results in the creation of a different codon. Because SNPs affect the genetic code of an organism, if the organism matures to reproduction age, these mutations can be passed down and become heritable and specific to a population.
Primer Design and Testing
Because SNPs are a point-mutation in the genetic code, in order to design a primer for the disease, one needs an already sequenced genome of a normal person. To create the forward primer, first one must find the position of the nucleotide that needs to change, then transcribe the preceding nineteen nucleotides to create a twenty nucleotide long strand with the changed-nucleotide at the end. To create the reverse primer, add two hundred to the position of the original code in order to reach the reverse primer, than write the bases from left to right of the bottom strand. To test the primer, one can copy both reverse and forward primer into a non-disease human genome sequence and check to make sure that there are no results. There must be no results since the program tests to check for non-disease where the SNP that was created is for a disease.
Citation Kitts A, Sherry S. The Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation. 2002 Oct 9 [Updated 2011 Feb 2]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 5. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21088/
BME 100 Fall 2017
