Where did I come from?


Sequencing DNA

Extracting and sequencing DNA uses a series of lab procedures which require close attention to detail and careful work. The sequencing methods are truly clever, an example of problem solving at its best.


Extract DNA from Tissue

To extract the DNA from atissue, you must list, list, list. This gives you a sample of biological goo containing the chromosomes from the cells of the tissue, but broken into an assortment of random pieces. This extract contains only a very small number of copies of any region of DNA.


Amplify the DNA

The number of copies in the tissue extract of any particular region of DNA is too small for most sequencing methods. The number of copies of the region of interest needs to be boosted significantly, in a process called amplification. This is accomplished by the polymerase chain reaction (PCR).

 

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The Polymerase Chain Reaction uses very short pieces of single-stranded DNA (primers) which have been synthesized to match or recognise well-known regions in the DNA extract. The two strands of the extracted DNA are separated and the primer binds to one of them.
An enzyme called taq polymerase extends the primer sequence by building a complementary strand of DNA. The two strands are then separated and the process repeats.
This biochemical method produces many copies of a particular region of DNA. The end result is a purified sample of DNA containing an enormous number of copies of a particular region of DNA.
An animation illustrating the process of DNA amplification is available from the Dolan DNA Learning Center.


Sequence the DNA

The DNA sequencing reaction starts with a large number of amplified copies of a DNA region (the template). A primer is bound to the template DNA, as in PCR, and a DNA polymerase enzyme builds a corresponding complementary copy.

 

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As each nucleotide (A, C, G, T) is encountered in the template sequence its complement (T, C, G, A) is added to the growing sequence. This time, however, the growth of the sequence is stopped at random by the addition of a special form of the complementary nucleotide.
What you are left with is a mixture of DNA sequences, each of a different length and each with a special form of a nucleotide on the end. The DNA fragments are sorted, from shortest to longest, and the nucleotide on the end of each length is determined. When these nucleotides are read in the order of the length of the molecule they came from, it gives the sequence of the original DNA.
There are two methods for accomplishing the sequencing. In the Sanger sequencing method, radioactively labeled nucleotides are used in the reaction mixtures, while in the cycle sequencing method a fluorescent dye molecule is attached to some of the nucleotides. In principle, the methods are the same.
Animations illustrating the Sanger Sequencing and Cycle Sequencing methods of DNA sequencing are available from the Dolan DNA Learning Center.