6/25/2006

Microwhatnows?

(Functional Genomics)

It's actually quite hard to read around genomics for any length of time without coming across the concept of microarrays. The problem is, no-one ever seems to explain what they are - you might as just well write "and then a miracle occurs". Now, after two google searches, six websites and a textbook, I've finally got my head round what they do. Read on, MacDuff!

Microarrays are used for sequencing DNA. As I hope to God you already know, DNA comes in four flavours, which pair up - an adenine molecule is always linked to a thymine molecule, a cytosine to a guanine, and vice versa. The resulting quaternary (four-element) code is copied by messenger RNA, transported to ribosomes, and there decoded as a protein molecule. It is, of course, fundamental to bioinformatics to know what these sequences of bases look like. However, these aren't exactly things that you can examine with the naked eye, so some scientific wizardry is necessary.

The way that microarrays sequence DNA is fairly simple. First, you'll need a tray with lots of holes in. Then you'll need to synthesise a bunch of different oligonucleotides. Oligonucleotides are one of many varieties of molecule that can "pair up" with DNA bases as if it was a second strand of DNA. Its critical feature, though, is that it is possible to detect whether or not it has paired up in this fashion with a DNA sequence. Your tray will need to contain lots of different oligonucleotide chains, and you'll need to keep track of which variety you put in which hole (mass production is your friend here).

Next, take your chunk of DNA. You'll need to make many many copies of it, but fortunately that's fairly easy to achieve (after all, cells do it all the time). Drop some into each hole.

This is the clever part. If the oligonucleotide in a given hole pairs up perfectly with some of the DNA you just dropped in, it will stick to it. If it doesn't, it won't stick under any circumstances. So, by using the detection technique alluded to earlier, you can tell whether the DNA sequence contains the subsequence that the oligonucleotide pairs to.

Once you've done all this, you'll have hundreds of bits of data - a long list of oligonucleotides that did stick, and a far longer list of ones that didn't. Most of these subsequences will overlap with each other, so you can "jigsaw" the complete sequence together.

For illustrative purposes, here's an example. Say you're using oligonucleotides of length 10 bases, and you want to sequence the following bit of DNA: TATACTTACGACCAG. Of course you don't actually know that this is its sequence, but when you drop it into the wells of the microarray the following pairings will occur:

TATACTTACGACCAG
ATATGAATGC

TATACTTACGACCAG
 TATGAATGCT

TATACTTACGACCAG
  ATGAATGCTG

TATACTTACGACCAG
   TGAATGCTGG

TATACTTACGACCAG
    GAATGCTGGT

TATACTTACGACCAG
     AATGCTGGTC


The wells with those oligonucleotides in will light up (literally - the detection process is usually based on fluorescence), and all the others will stay dark. Thus, you now know that the chunk of DNA contained the following subsequences and no others:
    TATACTTACG
    ATACTTACGA
    TACTTACGAC
    ACTTACGACC
    CTTACGACCA
    TTACGACCAG

The alignment of all these chunks is generally done by computer (in fact the entire process is generally automated) but, in this simple case, it doesn't take a genius to figure out what the overall sequence looks like.

Note: I'm in a fairly incoherent mood this evening, and I feel that may have fed over onto my blogging. If anyone has trouble figuring out what I'm on about in the above post, I'll make a special effort to fix it.

The rest of the time, you're on your own :P

2 Comments:

Blogger John Wendt said...

Not that I'm any kind of expert, but my impression is that arrays are used more for figuring out what genes are being expressed at a given time, than for sequencing a gene. You have to know the sequence beforehand in order to make the cRNA.

6/28/2006 10:38 pm  
Blogger Coalescent said...

You're almost certainly more accurate than I am. I really need to get a book on the subject.

6/29/2006 2:03 am  

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