An Online Introduction

to Advanced Biology


 Terms and Concepts 


SUBSITE 5 - Biochemical Analysis

CHAPTER TWO - Analysis of DNA


 Matching and Comparing Samples of DNA



The ability of biochemists to analyze samples of DNA has a short history.  For a few decades, analysis was restricted to manageable bits of DNA from high-volume samples (remember, in any sample of cells the DNA is a tiny fraction of whats in there).  Bits were obtained by using enzymes that could cut DNA at certain sequences.  These restriction enzymes (also called restriction endonucleases) are native to bacteria, where they are used to cut up DNA from viral invaders (the bacteria have protective proteins on their own DNA to keep the enzymes away from it).  Electrophoresis is used to spread the bits based upon size (and charge, but this is not as different among bits of DNA - can you guess why-?), producing long, banded smears rather than the fairly discrete bands common in protein analysis - there are too many different pieces to separate.  Then analysis could focus on the specific pieces.

In the Southern Blot technique, the bits of DNA are denatured (separating the strands) and transferred to nylon or specialized nitrocellulose paper using heat.  That paper can be tested for specific genetic sequences by applying bits of RNA carrying radioactive markers - the RNA will only bind to complementary DNA sequences.  Those radioactive bands can be easily identified with photographic film - just leaving the paper and film as a "sandwich" for a time will produce bands where the radioactive RNA has attached.  Until recently, the RNA probes had to be made using many cells doing some highly-active and specific process - they would be producing lots of RNA, enough to isolate and make radioactive probes from, to make the process-related proteins.  Now, the polymerase chain reaction discussed below allows the production of probes from single-stranded DNA.  There are also alternatives to the use of radioactive materials.

Using DNA in research was limited to samples with enough volume, rarely a feature of material left at a crime scene, and analysis in such areas as criminal forensics was very limited, up into the middle of the 1990s.


A Landmark Technique - the Polymerase Chain Reaction


As mentioned above, the options for testing DNA were limited by the amounts available - there were a few ways, including inserting DNA into bacteria, that allowed researchers to multiply a sample, but they were limited and time-consuming.  In the early 1980s, a researcher named Kary Mullis perfected a technique, the Polymerase Chain Reaction  or PCR, by which a small sample could be chemically magnified in a few steps.  One step involved heating the DNA to separate the strands, but this tended to degrade the DNA Polymerase used to make copies of the sample and copies of the copies.  Eventually, the process was stabilized by using a DNA polymerase taken from hot-spring bacteria (Thermus aquaticus, giving us "Taq" DNA Polymerase), which had evolved enzymes that would not denature at high temperatures.  The process continues to be protected by patent and usable only through a licensing agreement - a basic research tool that revolutionized many areas of biology and several folks bank accounts.

The basic process sounds simple, but of course its a bit trickier than it sounds.  Basically, a small DNA sample is added to a a mixture containing DNA primers and DNA polymerase.  The temperature is raised until the DNA in the sample separates into single strands (at about 94o - 96o C), then lowered (50o - 65o C) so the primers - short (20 - 50 bases) single strands of DNA set up to target particular stretches in the sample - attach to particular bits of single-strand sample.  At an intermediate temperature (around 72o C) DNA polymerase "fills in" the gap between beginning primers and end primers, making a copy of that section.  The heat goes up again, separating the strands, then drops, and now originals and copies are being copied, and then up, then down, through 20 to 30 cycles.  What had been a tiny sample is now millions of copies, usually more than enough for extensive analysis.

The PCR has made it possible to do genetic analysis on ancient DNA as well as the small residues from blood, hair, cell, and semen samples left at crime scenes.  It has been a quantum leap in genetic research techniques whose possibilities are still being explored.




 DNA Sequencing and Genome Mapping


It was decided in the 1980s that, although we were (and are) far from understanding what all of DNA was for, and we had isolated just a fraction of active genes, it would be useful if we had a "map," or total sequence, of human DNA, or our genome, and the Human Genome Project was born.  Techniques were improved, developed, and automated, and what looked like a decades-long project was essentially "wrapped" in 1993 with the last chromosome sequence published.  This is a "representative" genome, of course, and wouldnt match your own sequence exactly.

This process also uses pieces of DNA, cut from chromosomes, and primer strands from which stretches are polymerized.  The batches are separated into four mixtures, each of which stops polymerization at a different base type ( A, C, G, T).  The different strands are then analyzed and compared by electrophoresis.  It has really been the advances in the automation of this procedure that has sped the process along.  Now genomes have been determined for several important research organisms, and expanded to just organisms of interest.  When important genes are isolated in human diseases or in studies from lab organisms, a quick search can often find where those genes or related ones are in the vast quantity of human DNA.

Over the last few years, genomes have been determined for a number of different organisms.  Of course, the major model organisms have all been done, including Drosophila fruit flies (genetics),  C. elegans (development),  Arabidopsis (plant functions), yeast (cellular functions), and mice.  Other species of interest, like the apes, or dogs, or horses, have been done, as well as a growing list of disease organisms.

Human genome research has begun to look at both populations and individuals, with an eye toward targeting of treatments and analysis of risk that can be done for individuals.  Unfortunately, the technology is on the verge of exceeding anyones ability to analyze it - not enough folks in bioinformatics yet...

In theory, knowing a gene can lead to finding the proteins involved in the processes, which can lead to understanding their modes of action.  Sometimes this actually happens.



And, of course, the music video.

A report on the problems with forensics DNA tests, on mixed and old samples.






Terms and Concepts

In the order they were covered.

Restriction Enzymes / Endonucleases  
Electrophoresis of DNA  
Southern Blot Technique     
Polymerase Chain Reaction / PCR  
DNA Sequencing  
Genome Mapping  



Online Introduction to Biology (Advanced)

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