The small French town of Lourdes is perhaps best known for its masses of pilgrims who go there to seek the supposedly holy waters of a small grotto in search of miraculous healing. Of course, of the hundreds of millions of people who have visited Lourdes (9 million last year alone), there have only been a handful of "confirmed miracles", meaning if Lourdes' history were thought of as a drug trial, the FDA would have condemned miracles long ago.
But now, Lourdes is beginning to get a new reputation. One for a completely different kind of crazy than the usual delusional flocks of faithful: a woman in Lourdes, who thought she was possessed by the devil, stabbed her mother to death using a crucifix (the crucifix scene from The Exorcist comes to mind...).
She apparently told police "I had visions in a dream. I saw that I was the devil, that I was evil," and proceeded to beat her mother senseless with anything within reach before murdering her with a crucifix. She was promptly carted away to the loony bin.
This is what I don't get. Lourdes is filled to the brim with Catholic pilgrims every year. Ask any random assortment of pilgrims, and at least some are sure to tell you that "God has spoken to them" or that their faith is strong because "God had revealed himself" to them. Lourdes itself is a place that became famous after a teenage girl claimed to have seen the Virgin Mary herself. Yet, none of these people are put though psychiatric assessment.
Why is it that when someone claims to see or hear God or Jesus, it's taken as a divine event. Yet when someone claims to hear or see the devil, they're labelled as crazy and institutionalized? It's a complete double standard. Both are controlled by the same psychological phenomena: they're both simply delusions of an imaginary being.
One might make the claim that, as in the case above, those who think themselves under the control of the devil commit violent acts, but that would be ignoring the multitude of times throughout history that masses of people were murdered after some leader believed he had been told by God himself that it was a good idea. (George Bush and Iraq, anyone?)
It's time to stop treating Godly visitations as being sane.
Wednesday, 25 March 2009
Monday, 16 March 2009
The Kunkel Method, or How You - Yes, You! - Can Intorduce Any Mutation you Please into any Gene you Want!
In molecular biology, there are often occasions where you might want to introduce a specific mutation into a particular gene. Let's say you are examining a particular protein, and you want to find out how crucial a certain cystine residue is. You might want to change that cystine residue into, perhaps, a tyrosine residue, and observe how the protein behaves. You could do this by random mutagenesis and sifting through thousands of mutants until you find the one that you need, or you could do it a much quicker way.
The Kunkel method is one such way, and it is actually quite simple, both practically and theoretically. The first step is to clone the gene you want to mutate into whatever plasmid you choose to use. You can use whatever cloning procedure you wish. It doesn't really matter how you clone, just as long as you have a plasmid with your gene in it.
Next, the plasmid must be transformed into an E.coli strain that is ung- dut-. The dut gene encodes for dUTPase, an enzyme that prevents the bacteria from incorporating uracil during DNA replication (remember that uracil pairs with adenine but only in RNA. It is dUTPase that prevents uracil from being used in DNA by destroying all the cell's reserves of uracil during replication). A strain that lacks ung (dut-) will then randomly add uracil to your plasmid when it replicates. E.coli, however, have a backup mechanism in case dUTPase fails. This is uracil deglycosidase, encoded by the ung gene. Uracil deglycosidase cleaves out any uracil that has been added to DNA if dUTPase has been slacking on the job. Transforming into a strain that is also dut- will make sure the uracil in your plasmid stays there.
The next step is to design a primer that contains the region of the gene which you wish to mutate, along with the mutation you want to introduce. If you wanted to change a cystine to a tyrosine, then your primer would span the codon for cystine, but contain the necessary base pair changes to turn that cystine into a tyrosine. This primer will anneal to your plasmid when it denatures, even if the primer does not match it's target 100%. Once you isolate your uracil-containing plasmid, you can do PCR using your mutated primers to create hybrid plasmids: each plasmid will now contain one strand without the mutation and uracil bases, and another strand with the mutation and lacking uracil.
The final step is to isolate this hybrid plasmid and transform it into a different strain that does contain the ung gene. The uracil deglycosidase will destroy the strands that contain uracil, leaving only the strands with your mutation. When the bacteria replicate, the resulting plasmids will contain your mutation on both strands. In essence, you have completely replaced the original gene with your mutated version! You are then free to do as you wish with your new mutated gene.
The Kunkel method, however, is a bit outdated. Many bioscience companies offer kits that allow you to do site-directed mutagenesis even easier. Stratagene, for example, provides a kit that works as follows:
1) Transform your plasmid with gene of interest into any regular laboratory strain. Most strains will be dam+. The dam gene allows the bacteria to methylate the DNA in the plasmid (that is, they will add methyl groups to spots on the DNA. This is a mechanism that the bacteria has to allow it to determine what DNA is its own, and what DNA might be from an invading virus, since viral DNA will not be methylated).
2) Isolate the methylated plasmid, and do PCR with primers containing the mutation you wish to introduce. This will create hemimethylated plasmids: one strand (the one with the original gene) will be methylated and one (the one with your mutated gene) will not be methylated.
3) Add a small amount of the enzyme DpnI to the reaction mix. DpnI actively cleaves up methylated DNA. This will destroy the strands from the original plasmid and leave only your mutated strands.
4) PCR the remaining fragments to produce complete plasmids that contain your mutated gene.
And there you have it. Now you can mutate any gene you want in any manner you wish, and only take a day to do it. No more screening thousands of mutants! Huzzah!
The Kunkel method is one such way, and it is actually quite simple, both practically and theoretically. The first step is to clone the gene you want to mutate into whatever plasmid you choose to use. You can use whatever cloning procedure you wish. It doesn't really matter how you clone, just as long as you have a plasmid with your gene in it.
Next, the plasmid must be transformed into an E.coli strain that is ung- dut-. The dut gene encodes for dUTPase, an enzyme that prevents the bacteria from incorporating uracil during DNA replication (remember that uracil pairs with adenine but only in RNA. It is dUTPase that prevents uracil from being used in DNA by destroying all the cell's reserves of uracil during replication). A strain that lacks ung (dut-) will then randomly add uracil to your plasmid when it replicates. E.coli, however, have a backup mechanism in case dUTPase fails. This is uracil deglycosidase, encoded by the ung gene. Uracil deglycosidase cleaves out any uracil that has been added to DNA if dUTPase has been slacking on the job. Transforming into a strain that is also dut- will make sure the uracil in your plasmid stays there.
The next step is to design a primer that contains the region of the gene which you wish to mutate, along with the mutation you want to introduce. If you wanted to change a cystine to a tyrosine, then your primer would span the codon for cystine, but contain the necessary base pair changes to turn that cystine into a tyrosine. This primer will anneal to your plasmid when it denatures, even if the primer does not match it's target 100%. Once you isolate your uracil-containing plasmid, you can do PCR using your mutated primers to create hybrid plasmids: each plasmid will now contain one strand without the mutation and uracil bases, and another strand with the mutation and lacking uracil.
The final step is to isolate this hybrid plasmid and transform it into a different strain that does contain the ung gene. The uracil deglycosidase will destroy the strands that contain uracil, leaving only the strands with your mutation. When the bacteria replicate, the resulting plasmids will contain your mutation on both strands. In essence, you have completely replaced the original gene with your mutated version! You are then free to do as you wish with your new mutated gene.
The Kunkel method, however, is a bit outdated. Many bioscience companies offer kits that allow you to do site-directed mutagenesis even easier. Stratagene, for example, provides a kit that works as follows:
1) Transform your plasmid with gene of interest into any regular laboratory strain. Most strains will be dam+. The dam gene allows the bacteria to methylate the DNA in the plasmid (that is, they will add methyl groups to spots on the DNA. This is a mechanism that the bacteria has to allow it to determine what DNA is its own, and what DNA might be from an invading virus, since viral DNA will not be methylated).
2) Isolate the methylated plasmid, and do PCR with primers containing the mutation you wish to introduce. This will create hemimethylated plasmids: one strand (the one with the original gene) will be methylated and one (the one with your mutated gene) will not be methylated.
3) Add a small amount of the enzyme DpnI to the reaction mix. DpnI actively cleaves up methylated DNA. This will destroy the strands from the original plasmid and leave only your mutated strands.
4) PCR the remaining fragments to produce complete plasmids that contain your mutated gene.
And there you have it. Now you can mutate any gene you want in any manner you wish, and only take a day to do it. No more screening thousands of mutants! Huzzah!
Wednesday, 4 March 2009
Canadian Government Continues to Dig its own Scientific Grave
Our government continues to inch its way closer to being scientifically six feet under. The latest anti-science nonsense from Harper's regime: rejecting a motion to recognize Charles Darwin on his birthday and the marvelous theory he formulated.
According to the official records, MP Pierre Paquette rose to make the following request:
According to The Canadian Press, most of the "nays" came from the Conservative MPs (big surprise there), whereas the majority of votes were "yays" from the other parties. Unfortunately, Paquette was looking for a unanimous decision, so "mostly yes" wasn't good enough.
In other words, our government has officially rejected evolution.
Now, perhaps the wording has something to do with this. Including "unanimous" was probably a big mistake, but the bigger problem lies in calling evolution "the only proven and recognized scientific explanation for the origin of man." While this is technically correct - evolution IS the only scientifically accepted theory of man's origins and has been observed, tested and pretty much proved over and over again - politicians are not scientists. They are laypeople who have a cursory understanding of evolution at best and a complete ignorance at worst. Asking them to pass a motion firmly stating that evolution has been proven and is the only accepted theory in the scientific community, when they don't really understand the nature of the theory and its proofs, is a dumb idea. Even if the nay-sayers DID accept evolutionary theory, their lack of understanding of evolution would probably have prevented them from voting for the motion.
This article from Maclean's argues that the reason it was rejected was because the conservatives have decided that they are going to vote no on any motion presented by the other parties, which, if true, is incredibly petty.
Either way, our government just gave another "Eff You" to science.
A nod to Larry Moran for bringing this to my attention.
According to the official records, MP Pierre Paquette rose to make the following request:
Mr. Speaker, I seek the unanimous consent of the House to adopt the following motion: That the House acknowledge the 200th anniversary of the birth of Charles Darwin and the 150th anniversary of the publication On the Origin of Species by Natural Selection or the Preservation of Favoured Races in the Struggle for Life, which launched the theory of evolution, the only proven and recognized scientific explanation for the origin of man. I believe you will find unanimous consent for adoption of this motion.It was promptly rejected.
According to The Canadian Press, most of the "nays" came from the Conservative MPs (big surprise there), whereas the majority of votes were "yays" from the other parties. Unfortunately, Paquette was looking for a unanimous decision, so "mostly yes" wasn't good enough.
In other words, our government has officially rejected evolution.
Now, perhaps the wording has something to do with this. Including "unanimous" was probably a big mistake, but the bigger problem lies in calling evolution "the only proven and recognized scientific explanation for the origin of man." While this is technically correct - evolution IS the only scientifically accepted theory of man's origins and has been observed, tested and pretty much proved over and over again - politicians are not scientists. They are laypeople who have a cursory understanding of evolution at best and a complete ignorance at worst. Asking them to pass a motion firmly stating that evolution has been proven and is the only accepted theory in the scientific community, when they don't really understand the nature of the theory and its proofs, is a dumb idea. Even if the nay-sayers DID accept evolutionary theory, their lack of understanding of evolution would probably have prevented them from voting for the motion.
This article from Maclean's argues that the reason it was rejected was because the conservatives have decided that they are going to vote no on any motion presented by the other parties, which, if true, is incredibly petty.
Either way, our government just gave another "Eff You" to science.
A nod to Larry Moran for bringing this to my attention.
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