DNA Sequencing

There are several methods available to determine the actual sequence of nucleotides in a segment of DNA. One procedure uses specially altered nucleotides called dideoxynucloetides which have been made either radioactive or fluorescent.

When enough of the targeted DNA fragment (marker) has been amplified (multiplied) by PCR, the mix is put through a series of DNA sequencing reactions that are a variation of PCR. The amplified product from the PCR is added to a reaction tube containing the same Taq DNA polymerase used in the PCR, a primer that can hybridize at the desired location on only one complementary strand of the DNA (as opposed to both strands in PCR), and all four of the nucleotide bases (A, T, C, G.)

In addition, small amounts of fluorescence labeled dideoxynucleotides (A, T, C, G) are added to the mixture. Dideoxynucleotides are human-made nucleotides whose sugar component is slightly different from that of the nucleotides that make up DNA. (There is no OH on the 3' carbon.) Dideoxynucleotides can be picked up and added to a growing DNA chain. However, as a result of this structural difference another nucleotide cannot be added at its 3' end. Consequently, if one of the dideoxynucleotides is added to a growing chain of nucleotides, the strand will be terminated. Each dideoxynucleotide is labeled with a different fluorescent compound so that it will give off an identifying color in a laser beam.

After 20 - 30 cycles of the PCR heating and cooling, the resulting mixture will contain a series of fragments of different lengths depending on how many bases had been added to the chain before one of the dideoxynucleotides sneaked in and blocked further growth.

The mix of billions of short fragments from the sequencing reactions is loaded into glass capillary tubes that contain a gel solution that serves as a sieving matrix.

During electrophoresis, a voltage is created across the gel so that one end is made positive and the other negative. Since DNA is slightly negative, its fragments will move to the positive end of the gel. Not surprisingly, the different length DNA strands migrate at different rates and therefore separate from each other according to size. The smallest strand travels the fastest.

As each DNA fragment reaches the bottom of the capillary tube, its fluorescence-labeled end dideoxynucleotide is excited by a laser beam that is directed at the bottom of the tube. For an animation of the process of gel electrophoresis see: http://allserv.rug.ac.be/~avierstr/principles/electroani.html

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam. An automatic scanner records each wavelength of light and a computer generates an electropherogram with colored peaks representing each wavelength in the sequence it passes through the beam. The 5' terminal base (the dideoxynucleotide) of the shortest fragment (that moves the fastest) is the first base in the electropherogram.

http://cse.fra.affrc.go.jp/ksaitoh/pGEM-F50.gif

The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand.

At the left is a plot of the colors detected in one 'lane' of a gel (one sample), scanned from smallest fragments to largest. The computer even interprets the colors by printing the nucleotide sequence across the top of the plot.

Alternatively, the DNA fragments are separated on

a flat gel in a tray. The background of the title cell at the top of this page is a photograph of laser scan of a sequencing gel run in a tray.

Above is a tracing of an electropherogram of one STR tetranucleotide repeat (GATA) showing 21 repeats (count them) flanked by three repeats of GACA on the 5' end and one at the 3' end. (Primers specifically designed to match the Pre-flanking and Post-flanking sequences defined the segment to be amplified in the PCR reactions.)

The UCLA Molecular Biology Tutorial has a nice video animation of this procedure at http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html Click on the "video button" in the drop down menu under sequencing.

There is also an animation of the scanning and detection device to be seen at http://allserv.rug.ac.be/~avierstr/principles/scanani.html

Hit Counter