The DNA sequencer at New Mexico Tech is funded by the National Science Foundation

Introduction

Have you ever wondered how scientists were able to genetically analyze O.J. Simpson’s blood or the stain on Monica Lewinsky’s dress? The answer is all in their DNA.  Before DNA sequencing it was difficult to connect physical evidence to suspects in a court of law.  Prosceutors had to prove beyond "reasonable doubt" that the suspect in court was the same person who committed the crime.  Now with the use of modern DNA analysis, suspects can be linked tophysical evidence through DNA analysis. Scientists can take a small sample of DNA and amplify  specific regions which can then be sized or sequenced.  By comparing the size of the  of suspect's DNA to samples collected at the crime scene, guilt or innocence can be determined.   The same instrumentation that can accurately size DNA can be used to obtain the sequence of the four bases. 

DNA Sequencing in the past

In the past, DNA sequencing was done through a tedious method called polyacrylamide gel electrophoresis (PAGE).  The process requires DNA to be radioactively labeled, placed on a gel to which an electrical current is applied.  The DNA fragments move through the gel according to sizes; smaller nucleotides move the fastest and larger ones move more slowly.  The gel is then laid on an X-film, which is developed after adequate time is allowed for exposure.  Finally, after multiple rechecking of the sequence for errors, the scientist writes the DNA code by hand. 

The radioactive chemicals used to label the DNA strands can be dangerous.  Federal law requires that radioactive material be kept for ten half life cycles before they can be disposed of; sometimes this can take up to 900 days.  Storage of the radioactive material is costly and takes up valuable amounts of space in the lab.

In the past few years, genetic scientists have upgraded the process of DNA sequencing through computer technology.  The discovery of two processes has allowed scientists more access to the secrets of DNA.  The first, called Polymerase Chain Reaction (PCR) is a process that allows for millions of copies of sample DNA.  The other improvement in DNA sequencing is the creation of DNA sequencing machines that replace gel electrophoresis with capillary electrophoresis.  The sequencing machines are capable of reading and storing the data and result in data with lower percentage errors.    One such machine is the Applied Biosystems (ABI) Prism 310 Genetic Analyzer.

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is the process that allows large amounts of the sample DNA to be made for analysis.  When DNA replicates in the cell, it uses enzymes (chemicals that accelerate chemical reactions) to aid in copying and checking for errors throughout the process.  PCR uses the same enzyme that replicates DNA in a cell to make  millions of copies in a test tube (See Figure 1: Diagram of PCR).

Figure 1: Polymerase Chain Reaction

The first step in PCR is to “unzip” the double-stranded DNA, allowing the DNA polymerase to use the separate strands as a template.  In order for the DNA polymerase to copy DNA, two components are required: a supply of the four nucleotides (A, G, C and T) and a primer.  The primer can be labeled with a florescent dye so the DNA will be labeled.  Alternatively, the labeled nucleotides can be used.

Contained in the test tube are large amounts of the necessary materials for PCR.  The three steps of PCR process are carried out in the same test tube, but at different temperatures.  The first step is to heat the test tube to 90-95 ^BC for 30 seconds.  This heating causes the DNA to “unzip” by breaking the hydrogen bonds between the nucleotide bases.  In order for the DNA primer to anneal (bond) to the DNA, the test tube needs to be cooled to about 55 ^BC for 20 seconds.  The next step of the PCR process requires that the test tube contents be raised to 72 ^BC so that the DNA polymerase can create a new complementary DNA strand (See Figure. 1).    The strands are then labeled, so that the laser will cause each nucleotide to fluoresce a different color.  The new strand is called complementary because the DNA polymerase adds the complementary nucleotide (“A” bonds with “T,” “G” bonds with “C” and vice versa) as it copies the new strand (See Figure 2).

Figure 2: Complementary DNA Strands

This figure shows how A-T always pair together, C-G always pair together, and vice versa.

There are multiple advantages to the PCR process for genetic researchers.  First, the each cycle takes a total of two minutes.  In about three hours a scientist can produce over a billion copies of the desired DNA.  PCR is a critical tool in the Human Genome Project, in which the sequence of 90% of the human genome has been determined.  PCR can be used to identify  suspects in some crimes by amplifying and analyzing stains found at the crime scene.

Components of the ABI Prism 310 Genetic Analyzer

 The components of the Applied Biosystems (ABI) Prism 310 Genetic Analyzer are shown in Figure 3.  The DNA migrates through a capillary with a 50 micron bore.  Aligning the capillary with the sample tray is the most difficult step of the process because the capillary is very thin and must be aligned perfectly with a small dot on the sample tray. If this procedure is not done properly, the capillary will not be able to take up any DNA sample to be sequenced.  The sample tray can hold up to 96 test tubes to be tested at a time.

ABI Prism 310 Genetic Analyzer

The next component is the laser.  The laser allows the four nucleotides to radiate different colors of the visible light spectrum (See Figure 4: Laser Labeling).  The laser causes the dye on the nucleotides to illuminate a different color.  The camera then records these colors and sends the results to the computer for analysis.