ARMS

E.g. AAT

Vial A = normal; vial B = mutation

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 Repeat Sizing

E.g. Gilbert’s

Result

Grey bars indicate bins of which there are four (one for each repeat length). From
left to right the bins represent 5, 6, 7 and 8 repeat units.

The following repeat genotypes are not associated with Gilbert’s syndrome:
5-5, 5-6, 6-6, and 6-7. The following genotypes are associated with Gilbert’s
syndrome: 7-7, 7-8, and 8-8.

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 Taqman (Real time PCR)

E.g. HFE
Green = normal allele
Yellow = mutant allele
Result

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 Sequencing

E.g. Porphyrias

PCR but in the presence of dideoxyribonucleotides. This is just like regular DNA,
except it has no 3' hydroxyl group so once it's added to the end of a DNA strand,
there's no way to continue elongating it.

Now the key to this is that most of the nucleotides are regular ones, and just a
fraction of them are dideoxynucleotides. In dye-terminator sequencing, each of the
four dideoxynucleotide chain terminators (A, T, G, C) is labelled with a fluorescent
dye with different wavelengths of fluorescence and emission. Most of the time when
a 'T' is required to make the new strand, the enzyme will get a good one and there's
no problem. Most of the time after adding a T, the enzyme will go ahead and add
more nucleotides. However, 5% of the time, the enzyme will get a dideoxy-T, and
that strand can never again be elongated. It eventually breaks away from the
enzyme, a dead end product. Sooner or later all of the copies will get terminated by
a T, but each time the enzyme makes a new strand, the place it gets stopped will be
random. In millions of starts, there will be strands stopping at every possible T along
the way.

Gel electrophoresis can be used to separate the fragments by size and measure
them.

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In a large-scale sequencing lab, we use a machine to run the electrophoresis step
and to monitor the different colours as they come out. Since about 2001, these
machines (automated DNA sequencers) have used 'capillary electrophoresis', where
the fragments are piped through a tiny glass-fibre capillary during the
electrophoresis step, and they come out the far end in size-order. There's an
ultraviolet laser built into the machine that shoots through the liquid emerging from
the end of the capillaries, checking for pulses of fluorescent colours to emerge. We
don't even have to 'read' the sequence from the gel - the computer does that for us.

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Some other examples:

Disease Mutation Molecular Method

Sickle disease Single bp mutation ARMS

Taqman
RFLP if applicable

Thalassaemias Deletion (up to 3000bp) Gap PCR

Duchenne Muscular Large deletion Southern blotting

Dystrophy Arrays