Pick a primer set to target a particular gene fragment, such as a gene containing a known mutation (a single nucleotide polymorphism, or SNP), or look at a gene in another organism to learn about its genetic history and identify which species it comes from (DNA barcoding).
extract some of your genomic DNA from buccal cells
The human genome is a double helix formed by two complementary nucleotide chains
each human genome is broken into 23 pieces, called chromosomes
The genome is made up of four different nucleotides, adenine, cytosine, guanine, and thiamine. A-T and G=C can bind together- this is called “base pairing”.
because of base pairing, if you know the sequence of nucleotides on one chain of the double helix, you can infer the sequence of the complementary chain.
All of your body’s cells have a two complete copies of your genome (they are diploid)
(but: mature red blood cells do not have a nucleus or a genome, and reproductive cells only have one copy. They are haploid).
Each human genome is made up of around 3 billion base pairs. These encode around 20,000 genes.
Sequencing the first human genome took hundreds of scientists close to a decade and cost over 1 billion dollars (fact check).
Advances in sequencing technology (second-gen sequencing) have lowered the cost of genome-size sequencing
however, it still costs tens of thousands of dollars to sequence a human genome
Sanger sequencing, on the other hand, is an earlier sequencing technology that is very widely used. Sanger sequencing typically costs around $10 and is available as a service from a multitude of commercial sequencing companies. However:
Sanger sequencing requires a DNA to be relatively short in length (thousands of bases, or Kb, is ok), concentrated, and pure.
In other words, to use Sanger sequencing to sequence DNA, you must prepare billions of identical copies of the same short length of DNA you are interested in (fact check). Sanger sequencing will not work if all the copies of DNA that you prepare do not have the same sequence.
Polymerase Chain Reaction
The Polymerase Chain Reaction (PCR) can be used to make many copies of a desired DNA sequence. Amplifying a DNA sequence with PCR is one way to prepare DNA for Sanger Sequencing
PCR is accomplished with several special chemicals in a machine called a PCR thermocycler
Open up the cheek swab by tearing off the paper wrapper. Rub it and roll it on the inside of your cheeks 20 times.
side note: The swab looks like cotton, but it's actually a plastic polymer. The foam is heat-pressed to the the shaft of the swab instead of being glued to avoid potential interactions between the PCR mastermix we'll use later and glue.
Use one of the 4 pipets to transfer fluid from Tube A to Tube B until it is 1/2 full (3-4 transfers).
To use the pipet: grasp the bulb on top between your thumb and index finger. Hold the pipet vertically without any slant.
1. squeeze the bulb shut to displace air from the inside of the pipet
2. insert the tip of the pipet into the tube and look to make sure you have got it dipped inside fluid.
3. while keeping the tip immersed in the fluid, release your squeeze on the bulb. Fluid will be drawn up into the pipet tube
4. move the tip into the second tube (tube B). Squeeze the bulb harder and dispense all the fluid into the tube.
protip: don't press the pipet tip against the bottom of the tube when sucking up fluid or dispensing it or you may seal the tip shut. Instead, try to hold the tip at the bottom of the fluid but above the floor of the tube .
protip: if you get a bubble in the bottom of your tube after dispensing, you can use the empty pipet to suck up the air. Or you can close the lid of the tube and tap it against your table top to free up the bubble.
Using a NEW pipet, transfer one full drop from tube B into tube C.
be sure to hold the pipet vertically! it is calibrated to drop exactly 20 uL of fluid, but only when it is held vertically.
Tube C contains 180 uL of primers. Primers are short single-stranded pieces of DNA (20-30 base pairs long) that you use to target a specific range of DNA (usually 100 - 1000 bp long) for amplification with PCR.
After the transfer, close the lid of the tube. Tap the tube against your table top to totally dissolve the blue MasterMix pellet.
If little droplets form on the sidewalls of the tube while you tap it, try gently tapping to combine them all. It's important to have just one fluid droplet in the bottom of the tube before the next step.