The Basics of DNA Sequencing

SequencingWhy do we need to sequence our DNA?
Now that your DNA segment is amplified and purified, it’s time to read the genetic code or the sequence of nucleotides. Determining the order of the nucleotides in the piece of DNA you are studying is how you “read” the genetic code.

How do you sequence DNA?
There are many methods and the field is progressing rapidly, but many methods are based on dideoxy sequencing, which is also known as chain-termination sequencing or the Sanger method. It is very similar to PCR with a few exceptions: in addition to regular nucleotidetriphosphates (dNTPs: the letters A, T, C, G) the reaction includes a small amount of dideoxynucleotidetriphosphates (ddNTPs). The reaction adds ddNTPs to the growing chain just like regular dNTPs, except that once a ddNTP is added, the chain cannot grow any longer. The chain stops, or terminates, and a new chain is started in the next reaction.

When ddNTPs are added to a PCR, you get fragments of many different lengths instead of just one length. If you added only dd-Thymine to a PCR, then you know that all the fragments end in T. Typically, there are four different PCR reactions set up for each sequence: each reaction has a different ddNTP added to it. For example, if dd-Thymine is added to a reaction, you know that all the fragments will end in “T”. Then, if you know what the last letter of each fragment is, you can sort them by size and find the sequence by getting the last letter on each strand.

This method is very tedious to do by hand and you can only sequence short fragments. Modern techniques have mechanized the sequencing process for greater accuracy, ease, and the ability to read longer fragments. However, the basis for any chain-termination method is still the same.

Extreme Sequencing!
In 2003, an entire human genome (full set of genetic material) was sequenced. It took 13 years to sequence over 3 billion base pairs and the 20,000 known genes encoded by the human genome. Just in the past few years, sequencing technology has increased in speed and decreased in cost. Cutting edge processes, such as the 454 system, can sequence an entire human genome in three days. And sequencing will only get faster and less costly in the future! Thousands of organisms have been fully sequenced. Although many of them are bacteria and viruses, vertebrates such as the chicken, rat, orangutan, and giant panda have also been fully sequenced.

What does the sequence tell you?
It depends on what you are looking for! By comparing the sequence of a piece of DNA to others, you can determine how similar or different it is to the others. This information can be used for many different purposes, for example:

  • Forensics: identify the perpetrator of an unsolved crime by matching DNA samples from the crime scene to suspects
  • Medicine: determine if someone is at risk for a hereditary disease; sequence and identify viruses or disease-causing organisms
  • Zoology: calculate how much genetic diversity remains in a population of rare animals to see how at-risk they are
  • Agriculture: determine if crops have been contaminated by GMO plants in nearby fields
  • Microbiology: count the number of different species of bacteria that live in your intestines (tiny organisms like bacteria are often easier to identify by their genetic sequence than by looking at them)

How do you compare a sequence?
The increasing number of genes being sequenced combined with computer processing speeds has given rise to the field of Bioinformatics or the application of information technology to the field of biology and medicine. Many methods and algorithms exist for calculating how well one sequence matches with another, as the comparison gets more difficult as the sequences get longer.

[11] SC1.1 The student demonstrates an understanding of how science explains changes in life forms over time, including genetics, heredity, the process of natural selection, and biological evolution by relating the structure of DNA to characteristics of an organism.