A friend of mine, who actually reads my blog occasionally, was very interested in the idea of DNA sequencing, fascinated by the thought that DNA could just be created in companies and then shipped out when needed. He mentioned I should write a post on DNA sequencing.
I thought about it, and realised, with a daunting sense of dread, that actually I would have to do quite a bit of research before being able to write coherently about DNA sequencing. I know the general idea, but not enough to explain to someone who doesn't already have quite a good idea of whats going on. Luckily there already is a very clear and comprehensive explanation of it over at Genetic Interference:
Part One
Part Two
So I'll just add to the story a little by describing things from the point of view of me...the scientist actually ordering the DNA.
First I need to find out what I want. This requires a literature search. For example, when I started looking for my pigment colours, I went on a quick trawl through PubMed, looking for any genes that had been shown to produce colour in E. coli. The vio gene shown in this post is just one example, at the moment I also have a brown pigment, and (hopefully soon) two genes that make green and red as well.
The next stage is to find the actual DNA sequence. Usually it's in the PubMed paper, or in NCBI - which has a huge database of all proteins that people have registered. If it's very new research, you might have to email or phone the researchers. Once you have the DNA it's a good idea to double-check it as well...compare to homologous proteins or ones with similar domains. I used the MUSCLE comparison tool for this, simply because it's the one I'm most used to.
Once you're certain you have the right sequence for what you want, you contact a DNA synthesis company. Prices vary... as far as I can work out it varies from 20 (if you're REALLY lucky) to 50p per base pair. A smallish gene is usually about 1kilo-base pair(kb) just to give an idea of the scale of things. And the price tends to just up once you get over 1kb as well. The vio gene which we are getting for free is about 6kb long.
Then you send your sequence off to get made! You can have various options for synthesis (as I am just discovering). The codons (AAA, GGG etc) can be optimised for your organism - in the case of more than one codon (the three bases) coding for one amino acid, different organisms will prefer to use different codons. You can get restriction sites removed and added (for cutting and pasting DNA parts), and extra parts added to the gene, such as an area for the beginning of protein coding, or a degradation tag, which will cause the end product protein to break down (very useful if it's a long-living protein you want to get rid of quickly).
Larger genes get sent in bacteria, on little loops of DNA called plasmids to keep them replicating inside the bacteria. You grow your culture up, and then can extract your precious DNA from them. Small bits of DNA, like primers, just arrive as naked DNA, inside a little plastic vial, and can be made up to solution with water.
After synthesis, it's a good idea to get them sequenced as well... just to check you have the right stuff.
Field of Science
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Change of address1 year ago in Variety of Life
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Change of address1 year ago in Catalogue of Organisms
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Earth Day: Pogo and our responsibility1 year ago in Doc Madhattan
Showing posts with label DNA extraction. Show all posts
Showing posts with label DNA extraction. Show all posts
Lab Rat Working
Holidays are over :(
Image from here
I did not do the drawing, I hasten to add, but I do feel a bit like that. Sitting on top of seemingly impossible problems with a 'help meeee' sort of look.
At the moment I have decided to concentrate on revision because I am too tired (read lazy) to write my long essay. As my lever-arch file that contains the lecture notes is teetering on the brink of exploding, I've brought a book to write the revision notes in. So far I've filled in approximately half a page of it (NOT GOOD) but hopefully as it is a book rather than bits of paper I can do a NaNoWriMo thingy and try to fill up two pages a day.
Rather than, say, looking for pictures of rats on deviantart. heh.
Interesting Fact Of The Day: As there is now a large database of protein structures, many new structures can be determined by just looking at a DNA or protein structure and comparing it to the ones in the database. If you have the protein structure, you can also look at the individual amino-acids (the molecular building blocks that make up proteins) and using the knowledge of what kind of amino-acids they are (attracted to water, attracted to each other, acidic, polar, etc) make a reasonable guess as to the function.
This is one of those wonderful examples where science is very similar to trying to guess what the picture is on a half finished jigsaw puzzle (without looking at the box. In science there is no box). The more different techniques you use, the better you'll have an understanding of what the protein is, and of course some proteins are a lot easier to guess than others. A protein from a rat (say) with the exact same genetic code as a ribosome protein in a silverfish is easy. Another protein might have sections similar to the silverfish, but other bits that are more like bacterial membrane transporters, in whicch case you'll need a whole barrage of other techniques to figure it out.
Although if your DNA code is wrong to start with; due to contamination, procedural error, crossed communication wires or laziness, none of the fancy computer techniques will give any meaningful result. Nor will any of your experimental work, if you're recreated the protein from the genetic code you've been given.
And this does happen. There was a protein that developmental scientists were looking at a while back where (through no fault of the experimenter) the DNA mutated mid way through the growth-and-extraction process, which meant that everyone who was working on it (trying to find out where it migrated to in developing frog eggs) was working on a protein that didn't exist. Only one amino acid had changed, but it meant that this protein didn't migrate anywhere and all the researchers and poor little phD students were tearing their hair out about it.
Until someone finally decided to go back and check the sequence, essentially go through the whole tedious growing-frogs-and-taking-out-their-DNA-and-getting-the-right-bit-out all over again. They found that the bit of DNA they got the second time around was different to the one everyone else was working on. After that I think they did a couple more checks (well ... I hope they did. To be honest I wouldn't be surprised if they didn't) just to make sure they really had the right stuff, and yay! it migrated and some people wrote some papers and were happy about it.
Quick question: How long was the interval between the first attempt to get the correct DNA sequence and the second?
Answer: 20 years.
Oooops :)
To be fair to the scientific establishment though, there is a good reason for that. Science works on funding, and you just don't get funding for something that has Been Done Before. Another thing science works on is papers. The more papers you write, the more likely you are to get more jobs (and more funding) and you wouldn't realistically be able to publish a paper that just confirms someone else's work, not unless the work in question is starting to look very dodgy, and really needs conformation.
To be ever fairer to the scientists (especially as I am one), those twenty years working with the wrong protein were probably not an entire waste of time. They would have generated a lot of new techniques, and probably several people who were really good at tracking proteins by the end of it.
heh. still funny though.
20 years of the wrong thing...
Image from here
I did not do the drawing, I hasten to add, but I do feel a bit like that. Sitting on top of seemingly impossible problems with a 'help meeee' sort of look.
At the moment I have decided to concentrate on revision because I am too tired (read lazy) to write my long essay. As my lever-arch file that contains the lecture notes is teetering on the brink of exploding, I've brought a book to write the revision notes in. So far I've filled in approximately half a page of it (NOT GOOD) but hopefully as it is a book rather than bits of paper I can do a NaNoWriMo thingy and try to fill up two pages a day.
Rather than, say, looking for pictures of rats on deviantart. heh.
Interesting Fact Of The Day: As there is now a large database of protein structures, many new structures can be determined by just looking at a DNA or protein structure and comparing it to the ones in the database. If you have the protein structure, you can also look at the individual amino-acids (the molecular building blocks that make up proteins) and using the knowledge of what kind of amino-acids they are (attracted to water, attracted to each other, acidic, polar, etc) make a reasonable guess as to the function.
This is one of those wonderful examples where science is very similar to trying to guess what the picture is on a half finished jigsaw puzzle (without looking at the box. In science there is no box). The more different techniques you use, the better you'll have an understanding of what the protein is, and of course some proteins are a lot easier to guess than others. A protein from a rat (say) with the exact same genetic code as a ribosome protein in a silverfish is easy. Another protein might have sections similar to the silverfish, but other bits that are more like bacterial membrane transporters, in whicch case you'll need a whole barrage of other techniques to figure it out.
Although if your DNA code is wrong to start with; due to contamination, procedural error, crossed communication wires or laziness, none of the fancy computer techniques will give any meaningful result. Nor will any of your experimental work, if you're recreated the protein from the genetic code you've been given.
And this does happen. There was a protein that developmental scientists were looking at a while back where (through no fault of the experimenter) the DNA mutated mid way through the growth-and-extraction process, which meant that everyone who was working on it (trying to find out where it migrated to in developing frog eggs) was working on a protein that didn't exist. Only one amino acid had changed, but it meant that this protein didn't migrate anywhere and all the researchers and poor little phD students were tearing their hair out about it.
Until someone finally decided to go back and check the sequence, essentially go through the whole tedious growing-frogs-and-taking-out-their-DNA-and-getting-the-right-bit-out all over again. They found that the bit of DNA they got the second time around was different to the one everyone else was working on. After that I think they did a couple more checks (well ... I hope they did. To be honest I wouldn't be surprised if they didn't) just to make sure they really had the right stuff, and yay! it migrated and some people wrote some papers and were happy about it.
Quick question: How long was the interval between the first attempt to get the correct DNA sequence and the second?
Answer: 20 years.
Oooops :)
To be fair to the scientific establishment though, there is a good reason for that. Science works on funding, and you just don't get funding for something that has Been Done Before. Another thing science works on is papers. The more papers you write, the more likely you are to get more jobs (and more funding) and you wouldn't realistically be able to publish a paper that just confirms someone else's work, not unless the work in question is starting to look very dodgy, and really needs conformation.
To be ever fairer to the scientists (especially as I am one), those twenty years working with the wrong protein were probably not an entire waste of time. They would have generated a lot of new techniques, and probably several people who were really good at tracking proteins by the end of it.
heh. still funny though.
20 years of the wrong thing...Image from here
And why have you used 84 plates?
The thing about scientific protocols is that they are meant to be exact and precise. Every step must be explained concisely and there should be a reason for all the methodology.
For example, for every protocol you use, you should be able to answer random questions about why you did what you did at each step. Why was the bacteria incubated for four hours? Why was the temperature kept at 50 degrees? Why was the product stored on ice?
The answers to these questions should be sensible and scientific. Temperatures, amounts and incubation's are used to optimise reactions. Every step should be planned to get the best possible result in the most efficient way.
At the moment, we're growing phages on agar plates. At one stage of the protocol, we use exactly 84 plates to grow them. Two of the plates are controls and four are for dilutions but the fact remains that every time we use exactly 84 plates, no matter what we're doing.
The answer (as can probably be guessed) is not scientific in the least. Science is not a cold and clinical organised space, no matter how much scientists want it to be. It is a wild and crazy world full of human error and things going random and even more human error. The problems are not just scientific, they are also spacial and temporal; incubators are only so big, parts of the lab are only open during certain hours.
And the big jars we use for incubating will only physically fit 42 plates. We've tried to stuff more in but the lids won't shut. And while we have three of them in total the incubator is quite small and only fits two at a time.
42 x 2 = 84
Which wouldn't seem so bad if it weren't for the dilutions and the controls, because once you've put them in there's only room for 39 plates of actual phage sample per jar. 42 is at least a nice round number, but a protocol always looks a bit odd if it starts with the phrase '39 plates were taken...' It begs the question, 'why 39?' and the answer is, scientifically, faintly embarrassing.
Science would be a lot more precise if the world stopped getting in the way.
But far less fun =D
For example, for every protocol you use, you should be able to answer random questions about why you did what you did at each step. Why was the bacteria incubated for four hours? Why was the temperature kept at 50 degrees? Why was the product stored on ice?
The answers to these questions should be sensible and scientific. Temperatures, amounts and incubation's are used to optimise reactions. Every step should be planned to get the best possible result in the most efficient way.
At the moment, we're growing phages on agar plates. At one stage of the protocol, we use exactly 84 plates to grow them. Two of the plates are controls and four are for dilutions but the fact remains that every time we use exactly 84 plates, no matter what we're doing.
The answer (as can probably be guessed) is not scientific in the least. Science is not a cold and clinical organised space, no matter how much scientists want it to be. It is a wild and crazy world full of human error and things going random and even more human error. The problems are not just scientific, they are also spacial and temporal; incubators are only so big, parts of the lab are only open during certain hours.
And the big jars we use for incubating will only physically fit 42 plates. We've tried to stuff more in but the lids won't shut. And while we have three of them in total the incubator is quite small and only fits two at a time.
42 x 2 = 84
Which wouldn't seem so bad if it weren't for the dilutions and the controls, because once you've put them in there's only room for 39 plates of actual phage sample per jar. 42 is at least a nice round number, but a protocol always looks a bit odd if it starts with the phrase '39 plates were taken...' It begs the question, 'why 39?' and the answer is, scientifically, faintly embarrassing.
Science would be a lot more precise if the world stopped getting in the way.
But far less fun =D
When you don't know who to trust...
To determine how much DNA is present in our DNA extractions, we use a neat little machine called a Nanodrop. This tells us how much DNA is present by giving us the absorption spectrums at A260 and A280 Abs. As DNA absorbs ultraviolet, this tells us what concentration of DNA we have.
It also tells us how contaminated our sample is. The ratio of the 260/280 should ideally be between 1.8 and 2. Any lower and there is significant protein contamination, and higher and there's probably lots of salt or something in there.
Or so we thought. And so quite a few of the references and pages seemed to suggest. Until, of course, we got to the Wikipedia article. This tells us, in no uncertain terms, that it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio, and even provides a little table to show that. It provides a citation link as well, which I can't get to. I've tried Google and Pubmed until I went round in circles, but no one wants to give me that paper. At least not for free :(
So who do we trust? The wikipedia article has a solid-looking table, whereas in most of the other things we read it was just a throwaway line. On the other hand "most of the other things we've read" includes the instruction manual for the machine, which should know what it's talking about. And I have yet to read the wikipedia citation.
On reflection, I think I will disbelieve Wikipedia this time. At any rate, it hardly matters because we still don't actually have any DNA.
Ah well. At least I'm getting paid for it =D
It also tells us how contaminated our sample is. The ratio of the 260/280 should ideally be between 1.8 and 2. Any lower and there is significant protein contamination, and higher and there's probably lots of salt or something in there.
Or so we thought. And so quite a few of the references and pages seemed to suggest. Until, of course, we got to the Wikipedia article. This tells us, in no uncertain terms, that it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio, and even provides a little table to show that. It provides a citation link as well, which I can't get to. I've tried Google and Pubmed until I went round in circles, but no one wants to give me that paper. At least not for free :(
So who do we trust? The wikipedia article has a solid-looking table, whereas in most of the other things we read it was just a throwaway line. On the other hand "most of the other things we've read" includes the instruction manual for the machine, which should know what it's talking about. And I have yet to read the wikipedia citation.
On reflection, I think I will disbelieve Wikipedia this time. At any rate, it hardly matters because we still don't actually have any DNA.
Ah well. At least I'm getting paid for it =D
I'm in less of a sciency mood...
...because I've just started watching Firefly. wow. such amazing characters. Such amazing character interactions. Such an awesome mix of sci-fi and western without clogging it with unnecessary romance. Seriously though, is there any other sci-fi movie where the pilot is in a loving and stable marriage? Or where explosions in space happen to the accompaniment of awesome guitar music rather than actual scientifically-incorrect explosions?
On the lab front, we are busy troubleshooting. The DNA extraction technique is extracting tiny amounts of DNA and the task today is to find out why. Which means going through the whole process all over again (it takes about six hours) extracting aliquots every time we so much as pick up an eppindorf and try to find out where exactly our DNA is going.
...
They never have problems like this on Serenity.
On the lab front, we are busy troubleshooting. The DNA extraction technique is extracting tiny amounts of DNA and the task today is to find out why. Which means going through the whole process all over again (it takes about six hours) extracting aliquots every time we so much as pick up an eppindorf and try to find out where exactly our DNA is going.
...
They never have problems like this on Serenity.
DNA Extraction
Todays task is extracting DNA from our T4 phages that we've spent the last two weeks growing and nurturing and caring for. To make this easier, we're using a ready-made Kit called QIAGEN (which I keep trying to pronounce qui-gon :D ). QIAGEN basically provides all the material and gives an idiot guide as to how to use it, which means that Lab Rat's task of the day involves going through said protocol and working out what is actually happening in each step.
First though, another quick notice from the Department of the Very Obvious:
---
DNA Extraction
1.Grow the phages on agarose gel. Agar gel is a gelatenous substance from seaweed that contains both agarose and agaropectin. Agaropectin contains lots of acidic sidegroups (containings sulfur and various other things like pyruvate). Agar is usually used for gels as it's cheaper, but for DNA extraction you can't risk any of the acidic agaropectin loitering around as it stops the extraction enzymes from working. Instead you use just pure agarose.
2.Isolate phages. This is done by peeling off the top layer of agarose and using a centrifuge to spin it down to the bottom, leaving the phages in the supernatant (the liquid left behind after centrifuging). Push the supernatant through a couple of filters to remove anything else (bacteria mostly) and you're left with a pure phage solution.
3.Now the DNA extraction can start. The first instruction in the kit is 'add buffer L1'. Buffer L1 is a clever mixture of various different enzymes and buffer solutions which breaks down any bacterial RNA or DNA that might be left in your solution.
4.Next buffer L2 is added. This precipitates out the phage particles; essentially it clumps all the bacteriophage's together making them easier to extract.
5.Centrifuge to collect the phage. The centrifuge is the big fast-spinning machine that pellets all the phages down into a neat little, well, pellet. Very useful machine, and it would be even more useful if ours worked properly :(
6.Resuspend the phage in buffer L3. I suspect this is just a growth medium, to turn the phage pellet into a phage suspension.
7.Add buffer L4. Buffer L4 contains the well known SDS (which comes up very often in various extractions). SDS is a detergent which essentially breaks all the proteins down. As viruses pretty much just consist of proteins and DNA this means that the only whole thing left in the test-tube at this point is the DNA. Still quite a way to go though, so here's a diagram of a centrifuge:
8. Add buffer L5, mix and centrifuge. It does not actually say, but i suspect L5 is precipitating the proteins that have just been cut up. The centrifuge will then pellet the proteins down to separate them from our DNA.
9. Equilibrate a QIAGEN-tip 20 by adding buffer QBT. This is where we hit the Magic Event Horizon (MEH) at top speed. I have no idea what is in the buffer or exactly how a QIAGEN-tip works, which is probably a good thing for copyright in general. You can get to QIAGEN to find out more about it by going here: QIAGEN
10. Allow the residue from step 8 to flow through the QIAGEN tip. One thing I do know about the tips is that they contain resin. The resin traps the DNA on it allowing the rest of the phage to wash through (essentially this will just be any liquid medium, as all the proteins have technically been removed by the centrifuge in step 8)
11. Wash the QIAGEN tip with buffer QC. This washes the DNA (which is trapped on the filter) removing any last impurities.
12. Collect DNA with buffer QC into a clean tube. The QC in some way (Magical Event Horizon fast approaching) allows the DNA to flow through the filter and into the new tube. Finally the DNA! We now (should) have a solution containing nothing but phage DNA. Just got to collect it.
13. Precipitate the DNA by adding isopropanol. Isopropanol is another old favourite, it just clumps DNA, making very very hard-to-see pellets. You've probably guessed by now but the next stage is: centrifuging, to collect the pellet.
14. Wash pellet with ethanol. Ethanol removes any residual salt as there will be some magnesium and various others in the DNA (DNA has an overall negative charge due to the phosphate backbone which collects positively charged salts). Washing involves adding ethanol, shaking very gently and then (surprise surprise) centrifuging to pellet the DNA again and remove all the ethanol.
15. Allow DNA to dry and redissolve in buffer. Buffer just keeps the DNA happy and stops it disintegrating.
And that's it! We now have a little glass bottle containing a solution of pure DNA.
Science really is just like cookery =D
First though, another quick notice from the Department of the Very Obvious:
- Do not try to do DNA extractions when hung-over
---
DNA Extraction
1.Grow the phages on agarose gel. Agar gel is a gelatenous substance from seaweed that contains both agarose and agaropectin. Agaropectin contains lots of acidic sidegroups (containings sulfur and various other things like pyruvate). Agar is usually used for gels as it's cheaper, but for DNA extraction you can't risk any of the acidic agaropectin loitering around as it stops the extraction enzymes from working. Instead you use just pure agarose.
2.Isolate phages. This is done by peeling off the top layer of agarose and using a centrifuge to spin it down to the bottom, leaving the phages in the supernatant (the liquid left behind after centrifuging). Push the supernatant through a couple of filters to remove anything else (bacteria mostly) and you're left with a pure phage solution.
3.Now the DNA extraction can start. The first instruction in the kit is 'add buffer L1'. Buffer L1 is a clever mixture of various different enzymes and buffer solutions which breaks down any bacterial RNA or DNA that might be left in your solution.
4.Next buffer L2 is added. This precipitates out the phage particles; essentially it clumps all the bacteriophage's together making them easier to extract.
5.Centrifuge to collect the phage. The centrifuge is the big fast-spinning machine that pellets all the phages down into a neat little, well, pellet. Very useful machine, and it would be even more useful if ours worked properly :(
6.Resuspend the phage in buffer L3. I suspect this is just a growth medium, to turn the phage pellet into a phage suspension.
7.Add buffer L4. Buffer L4 contains the well known SDS (which comes up very often in various extractions). SDS is a detergent which essentially breaks all the proteins down. As viruses pretty much just consist of proteins and DNA this means that the only whole thing left in the test-tube at this point is the DNA. Still quite a way to go though, so here's a diagram of a centrifuge:
8. Add buffer L5, mix and centrifuge. It does not actually say, but i suspect L5 is precipitating the proteins that have just been cut up. The centrifuge will then pellet the proteins down to separate them from our DNA.9. Equilibrate a QIAGEN-tip 20 by adding buffer QBT. This is where we hit the Magic Event Horizon (MEH) at top speed. I have no idea what is in the buffer or exactly how a QIAGEN-tip works, which is probably a good thing for copyright in general. You can get to QIAGEN to find out more about it by going here: QIAGEN
10. Allow the residue from step 8 to flow through the QIAGEN tip. One thing I do know about the tips is that they contain resin. The resin traps the DNA on it allowing the rest of the phage to wash through (essentially this will just be any liquid medium, as all the proteins have technically been removed by the centrifuge in step 8)
11. Wash the QIAGEN tip with buffer QC. This washes the DNA (which is trapped on the filter) removing any last impurities.
12. Collect DNA with buffer QC into a clean tube. The QC in some way (Magical Event Horizon fast approaching) allows the DNA to flow through the filter and into the new tube. Finally the DNA! We now (should) have a solution containing nothing but phage DNA. Just got to collect it.
13. Precipitate the DNA by adding isopropanol. Isopropanol is another old favourite, it just clumps DNA, making very very hard-to-see pellets. You've probably guessed by now but the next stage is: centrifuging, to collect the pellet.
14. Wash pellet with ethanol. Ethanol removes any residual salt as there will be some magnesium and various others in the DNA (DNA has an overall negative charge due to the phosphate backbone which collects positively charged salts). Washing involves adding ethanol, shaking very gently and then (surprise surprise) centrifuging to pellet the DNA again and remove all the ethanol.
15. Allow DNA to dry and redissolve in buffer. Buffer just keeps the DNA happy and stops it disintegrating.
And that's it! We now have a little glass bottle containing a solution of pure DNA.
Science really is just like cookery =D
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