Concept 23 A gene is a discrete sequence of DNA nucleotides.
Fred Sanger outlines DNA sequencing.
I'm Fred Sanger. In the '50s I was the first to determine the sequence of amino acids in a protein. I showed that the 51 amino acids of the insulin protein are arranged in a specific order. I'm Fred Sanger. In the 50's I was the first to determine the sequence of amino acids in a protein. I showed that the 51 amino acids of the insulin protein are arranged in a specific order. Since the genetic code determines the order of amino acids, the DNA sequence is colinear with the amino acid sequence. However, knowing the amino acid sequence of a protein does not tell us the exact nucleotide sequence of its gene. Remember that the genetic code is redundant – more than one codon can code for an amino acid. For example, here are the redundant codons for these amino acids. In the early '70s, I developed a method to determine the exact sequence of nucleotides in a gene. At about the same time, Alan Maxxam and Walter Gilbert developed a different sequencing method. As it turns out my "chain termination" method is most commonly used in labs today. My method was based on Arthur Kornberg's earlier work on DNA replication. Remember that a new DNA strand is synthesized using an existing strand as a template. Here, I am using poly-thymine DNA as an example. The 5' carbon of an "incoming" deoxynucleotide (dNTP) is joined to the 3' carbon at the end of the chain. Hydroxyl groups in each position form ester linkages with a central phosphate. In this way, the nucleotide chain elongates. The key to my sequencing method is the peculiar chemistry of dideoxynucleotides (didNTP). Like a deoxynucleotide, a didNTP is incorporated into a chain by forming a phosphodiester linkage at its 5' end. However, the didNTP lacks a 3' hydroxyl group (OH) necessary to form the linkage with an incoming nucleotide. So, the addition of a didNTP halts elongation. I set up four separate reactions – one to provide sequence information about each of the nucleotides. Each reaction contained: template DNA, a short primer (about 20 nucleotides), DNA polymerase, and the four dNTPs (one radioactively labeled). Then I carried out a sequence of steps. Let's focus on the adenine (A) terminator reaction. First, the DNA is denatured into single strands at near-boiling temperature. At high temperatures, the kinetic motion of the DNA molecule disrupts the weak hydrogen bonds that join complementary DNA strands together. When the temperature is lowered, the primer binds to its complementary sequence in the template DNA. Kornberg's earlier work showed that a primer is needed for DNA polymerase to start replication. The DNA polymerase makes no distinction between dNTPs or didNTPs. Each time a didNTP is incorporated, in this case did ATP, synthesis is "terminated" and a DNA strand of a discrete size is generated. In this sequencing example, the didATP (purple) has terminated the reaction. The dATP happens to be the radioactive tracer but this has no effect on elongation. Because billions of DNA molecules are present, the elongation reaction can be terminated at any adenine position. This results in collections of DNA strands of different lengths. The same is true for the other three terminator reactions. Each reaction is then loaded into a separate lane of a polyacrylamide gel containing urea, which prevents the DNA strands from renaturing during electrophoresis. Ionized phosphates give the DNA molecule a negative charge, so DNA migrate toward the positive pole of an electric field. The movement of DNA molecules through the polyacrylamide matrix is size dependent. A blue dye that also migrates to the + pole is added to the samples to track the progress of DNA in the sequencing gel. The dye runs just a little faster than the smaller fragments of DNA. Over the course of electrophoresis, shorter DNA molecules will move further down the gel than larger ones. Millions of terminated molecules of the same size will migrate to the same place and "band" in the gel. After electrophoresis, the gel is sandwiched against X-ray film. The radioactive adenine in the synthesized DNA emit beta particles that expose the film, making a record of the positions of DNA bands in the gel. The sequencing gel is then "read" from bottom to top. The sequence of bands in the various terminator lanes gives the sequence of nucleotides in the template DNA. A typical sequencing reaction will yield 200-500 base pairs of readable sequence.
At the time Fred Sanger was working on a method to sequence DNA, many scientists were trying to sequence RNA. They believed that since RNA was smaller, it should be easier to sequence.
It is technically more difficult to sequence RNA than DNA. Why do you think this is true?