Take me to the Steps

The goal

sequence a small fragment of your own genome.

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).

The plan

  • extract some of your genomic DNA from buccal cells
  • amplify a fragment of your genomic DNA using PCR
  • mail the DNA to our DNA sequencing center
  • register your sample at http://cofactorbio.com/claim
  • get your results in 2-5 days.

The details

Genomics 101

  • 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

  • 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
  • We’ll get into the details below.

Project Steps

arrange your tubes in the tube holder.

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.

open up Tube A, the genomic extraction buffer (you can leave the swab in your mouth to free up your hands).

dunk the swab (now with your cheek cells) into the buffer and roll it around on the inner walls 10 times. Squeegee excess buffer from the foam tip of the swab as you pull it out if you can.

discard the swab. It’s just got your dna on it and some proteins and salts. Safe to throw away.

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.

Transfer Tube B to your incubator or thermocyler. Incubate the tube at 65 C for 2 minutes

this incubation will activate the enzymes in the genomic extraction solution. They will break down the cellular material in your saliva sample, leaving behind genomic DNA.

If you get air bubbles in your pipet, dispense the fluid back into tube B and try again. It takes a little practice to avoid bubbles.

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.

Dip a new pipet into tube C and move it up and down (without squeezing the bulb) to mix the primer solution. Move up and down 10 times or so.

Now transfer 1 full drop into Tube D, in the same way as in step 5 (keeping the pipet vertical). You are mixing your genomic DNA & primers with PCR “MasterMix” (the blue pellet in Tube D).

PCR MasterMix contains a mix of free DNA base pairs, heat-stable DNA polymerase to copy your DNA using the primers, and cofactor ions like magnesium to help the polymerase work.

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.

Transfer Tube D to your PCR thermocyler. Run the following PCR thermocycling program:

1) 95 C for 5 min

2) 30 cycles of:

i. 95 C for 30 sec

ii. 55 C for 60 sec (see your specific instructions for different primer annealing temperatures)

iii. 72 C for 60 sec

3) 72 C for 5 min

Hold temp at 4 C

After the PCR program completes (90 min), use the last pipet to transfer the entire contents of tube D into the the DNA Sequencing Tube, (Tube E).

Be careful not to suck the amplified DNA from tube D too high into the pipet – it’ll be hard to get out. Try and keep the fluid near the tip.

It’s ok to have a little extra left over in the pipet after your done.

You’re almost done! Replace the cap on the DNA sequencing tube and seal it in the pre-addressed envelope. Drop this in the mail when you get a chance.

You’re mailing your amplified DNA to our DNA sequencing center, where it will be Sanger Sequenced by robots. When the robots are done, they’ll email you with the results via our website.

Last step: got to cofactorbio.com/claim and register your kit, using the serial number included with your labels and instructions.

This step is necessary for us to return your sequencing results to you via email.

All Done!

Conclusion

The conclusion