Lecture26 - BIS101 Lecture 26 DNA Sequencing You want to...

BIS101
Lecture 26
5/31/11
DNA Sequencing : You want to know the entire sequence of the gene you are interested in.
Variations of the chain termination method of DNA sequencing introduced by Sanger has been
automated and is being used to sequence whole genomes, such as the human genome. The
method is described in Figure 11-18 in 8 th ; Figure 20-16 in 9 th ; Figure 10-18 in 10 th edition. The
use of dideoxynucleotides results in chain termination during DNA synthesis. Spiking 4
reactions, each with one dideoxynucleotide (ddNTP), at concentrations that lead to random chain
termination, leads to a ladder of different fragments, each terminating with the specific
nucleotide. High resolution gel electrophoresis, that can resolve DNA fragments that have a
single nucleotide difference in length, are used to determine the size of the fragments. Coupled
with the knowledge from which reaction the fragment derived (ddA, ddC, ddG, ddT), one can
easily derive the DNA sequence of the original DNA. The fragments are visualized by either
radioactively labeling the primer or one of the dNTP nucleotides.
Present day DNA sequencing is automated using ddNTPs that each have a different
fluorescent label (Figure 11-19 in 8th; 20-17 in 9th; 10-19 in 10th). Therefore, what use to be
four separate reactions can now be accomplished in a single reaction using four different
fluorescent dyes. The DNA sequence is deduced from fluorescence profiles shown in the figure.
The whole process is automated. Currently, new technologies are being developed to sequence
more DNA at less cost. These include 454 sequencing that allows for detection of sequencing by
synthesis using chemiluminescence detection.
You should understand the principles behind DNA sequencing. In particular, realize that
these rely on the process of Watson-Crick base pairing rules and use the enzymes we
previously discussed with DNA replication.
Polymerase Chain Reaction = PCR: We saw a movie that described in some detail the
principle of the technique. I posted Handout31 on the web also describing PCR. Mullis won the
Nobel Prize in 1993 for development of PCR. This technique has revolutionized molecular
biology. This allows for the in vitro synthesis of almost any DNA molecule. What you need:
1) target DNA – you only need a small amount of DNA, for instance, a single cell
2) set of primers – these are short stretches of single stranded nucleotides that are
synthesized by organic chemistry that are homologous to your gene of interest. You
design them to base pair with the sequence you are interested in (which means you
need sequence information).
3) DNA polymerase called Taq polymerase because it is from a thermophilic (heat
loving) organism – Thermophilus aquaticus . This organisms lives at very high
temperatures so that the polymerase survives multiple rounds of heating.