Re: [DIYbio] Re: A simple project for PCR first-timers

[Cory Tobin]

> Forgive me if this is foolish, but does the Y within the primer sequence
> correspond to thymine or cytosine?

Yeah, the Y means a mixture of C and T. Most primer manufacturers
allow you to mix bases. Comes in handy sometimes.


> If so, will the primers contain
> a mixture of each sequence??

Correct. Ideally there will be two different oligos in equal proportions.


-cory

[Chuckles]

Makes sense! Thanks! 

Have you made any progress with step 4?

[abhay]

Interesting, Do you know, if human sex determination can also happen through PCR. Are there other species whose sex could be determined this way?

On Wednesday, June 20, 2012 8:40:50 AM UTC+2, Meow-Ludo wrote:

Beginners PCR Project

@DIYbio'ers - Please feel free to edit and revise this to make it usable by people that are keen to try out PCR. I would love to get it to a finished stage and then publish it as a PDF, as I saw a lot of posts from people that are keen to get into using PCR but don't know where to start. This could provide a way for people that have just built an openPCR to get started without too much fiddling around. Let me know what you think.

This practical will allow you to use PCR to identify the sex of birds. Most birds can be sexed this way due to a difference in the length of an intron (unexpressed 'spacer' section on DNA that is often highly variable) in a gene carried on the sex chromosomes.

In humans the heterogametic individual is male (XY), whilst the homogametic is female (XX). In birds this is reversed and the sex chromosomes are Z and W. Males will be ZZ and the females ZW.

By amplifying these genes and running them on a gel, the products can tell us if two Zs or ZW is present as the Z and W products are of different sizes. If there are two bands on the gel, the bird is female and conversely one band means it is male. This is really useful as the experiment has an inbuilt positive control (ie, no bands means your PCR didn't work).

This is a really good way to start in PCR as it is practical and well researched, taking out some of the hard work. In Australia companies charge around $15AUD per bird. If you shop around your costs for each reaction could well be under $5AUD per reaction making it a good little first business to fund your lab. Also, there are lots of brids around to experiment on, and a number of papers suggest approaching a local pet shop and asking if they want their birds sex tested for free. This is really good because it puts you in touch with customers that might like the service, but if you stuff up any of the reactions you haven't really lost anything.

For anyone not familiar with scientific articles, feel free to ask any questions you might have as I have tried to design this so that biohacking groups ca use this as an introduction to genetics. The format will be similar in structure to a scientific article.

Procedure:

1. Collect some chest feathers from some birds. Parrots are preferrable as this test works very well with them. This test will not work with rattites. Rattites are bascially anything that looks like an Emu. This may not be too much of a problem outside Australia/NZ. You will need only one or two feathers, plucked from the chest. This is because these feathers will have a very small amount of skin at the base. This should not be painful to the bird. Do not be tempted to collect them from the bottom of the cage as these feathers will not have enough viable DNA to serve as a template for the amplification carried out in the PCR. If this is your first time doing something like this you may want to collect 4-5 feathers per bird so that you can repeat the experiment if something goes awry. REMEMBER TO LABEL THE BIRD AND THE BAG. Once you have left the pet shop their may be no way for you to correlate your results to animal that they came from. Scientists often use a lab book to record everything they do during an experiment and this can be really useful. You may want to assign a code to each bird (BUDGIE-CAGE-1-GREEN) or take a identification from an ankle tag. It is the base of the feather that will be used so take care not to touch it or let it touch anything else as it become contaminated. Also, make sure that all the samples are kept individual from each other.

2. OPTIONAL: Storage. If you wish to keep the feathers for a while for some reason, say as not being able to immeditaely do the procedure, cut off the base of the feather where it was closest to the bird. This should have the DNA on it as either a tiny bit of blood or some tissue. Put this in a microcentrifuge tube with some high strength ethanol (>70%) until it is immersed. This can be stored at room temperature for a long time (years or more).

3.Extract the DNA. This may require a bit of testing to get it down pat. There are a lot of guides on the net to showing you how to do this but I am going to write a method that will work. You should be able to adapt this using more readily available or safe materials. Where possible I have tried to include reagents that eare easy to obtain and non-toxic. Make sure to read material safety data sheets (MSDS) for any reagents you use for specific safety requirements for working with them as products may vary in their requirements. These are found on the website that you obtain them from. If you are getting reagents from friends or eBay, the person supplying them should have these. The procedure I suggest is below. I haven't tested this yet, but will update it when I have. If anyone is keen to help out here it would be greatly appreciated.

1. Using a sterile blade transect the shaft of the feather (the end the grows from the bird) so that the internal section is open.

2. Put this into a 1.5ml microcentrifuge tube. Add to the tube:

3. Pick the protocol most relevant to you:

Home protocol (this is if you can't get access to lab grade reagents) 

• A detergent to bust open the cells. Ordinary detergent should work OK (just a tiny little drop). 
• If you used the detergent, you will need to add a little bit of table salt (just a little pinch) and a proteinase. Some pineapple or papaya juice should work, but others have suggested using purified bromelein.
• put the tubes, sealed, in some warm water (50°C) for 3-5 hours.

Lab protocol (if you have access to lab grade reagents and instruments)

•  make a lysis buffer in bulk (100ml of 1 M Tris, 200 ml of 0.5 M EDTA, 2 ml of 5 M NaCl, 100 ml of 10% SDS) 
  
• add 180ul of the lysis buffer to your microcentrifuge tube
• add 25 μl of 100 mg/ml DTT (dithiothreitol) and 20 μl of 10 mg/ml proteinase K 
• put the tubes, sealed, in some warm water (50°C) for 2-5 hours. 
  

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4. Ethanol Precipitation

I need a bit of help getting this stage done. I am used to working with phenol/chloroform but I am having troubles working out the best way to do this just using ethanol. Any help would be appreciated.

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Now that you have your DNA you can move onto the amplification stage. You will need some primers to select the region you wish to look at (in this case the CHD gene will be amplified) and all the things needed for replicating DNA. These are: a polymerase (copy machinery), dNTPs (building blocks of DNA), Mg2+ (helper of polymerase) and your template DNA and primers.

Make up a solution called a master mix. You may purchase this ready made, but here is what you need if you are making it yourself:

This is the volume per tube. Multiply this to get the total volume you need to prepare.

• 1.5 μl of 10x PCR buffer
• 1.5 μl of 25 mM MgCl2
• 100 ng of each primer  P8 (5’-CTCCCAAGGATGAGRAAYTG-3’) and P2 (5’-TCTGCATCGCTAAATCCTTT-3’)
• 200 μM of each dNTP
• 0.5 U Taq DNA Polymerase
• 250 ng of DNA sample ( This could be a bit tricky to work out if you don't have access to a spectrophotometer)

For the PCR reaction P8 (5’-CTCCCAAGGATGAGRAAYTG-3’) and P2 (5’-TCTGCATCGCTAAATCCTTT-3’) primers were used. These can be purchased from a scientific supplier. They bracket a section of DNA and specifically amplify just the section between them. They should be around the $10 mark. You need about 10-100ng of primers per reaction. The primers should come in such a quantity that you can perform hundreds of reactions with them. Ask your supplier about what form they come in as you may need to resuspend them if they come freeze-dried.

Program your PCR for amplification with the following:

• a denaturing step at 94 ºC for 1 min 30 s
• 35 cycles of 95 ºC for 30 s, 52ºC for 30 s, and 72 ºC for 30 s
• final elongation at 72 ºC for 5 min. PCR products were visualized on 5% agarose gel stained with GelRed or other safe DNA stain.
  

This protocol borrows heavily from:

Sex Determination by CHDW and CHDZ Genes of Avian Sex Chromosomes in Nymphicus hollandicus, Harun CERIT, Kozet AVANUS, 2007.Turk. J. Vet. Anim. Sci.

http://www.protocol-online.org/biology-forums/posts/18660.html 

Molecular techniques for sex identification in birds, ANNA DUBIEC and MAGDALENA ZAGALSKA-NEUBAUER. BIOLOGICAL LETT. 2006, 43(1): 3–12 (PDF)

[Meow-Ludo]

Yeah I have decided to use an EDNA kit. They work out really cheap (around $1 per extraction). If these aren't available where you are I would be happy to send them to you.

I am buying a pack of 100 which is the smallest amount they sell them in I believe. They are going to make everything as food-safe as possible too.

[Meow-Ludo]

Yes there are other species that can be sexed this way. Basically find a gene that is of different length (due to increase in highly variable regions) or find an oligo that binds different genes in male and female. There are more than likely a number more methods that could be used to do this.

I am really keen to get a sex test for pythons. DNA can be extracted from their sloughed skins and there is a demand for this service in Australia. If anyone is keen to help me with the project, let me know. The burmese python genome has been sequenced, so that might be a good place to start.