General Biology 2 - Analysis of DNA

Biology - Molecules and Cells

SECTION 5

CHAPTER 3 - DNA-Based Techniques

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 what's 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.

More on restriction enzymes.

Image.

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 developed 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.

Southern blot (video animation).

Protocol.

Variation: Northern Blot.

Variation: Western Blot.

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 1980's, 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.

PCR animation.

Another animation.

Making best use of Taq polymerase.

The basic process sounds simple, but of course it's a bit trickier than it sounds. Basically, a small DNA sample is added to a mixture containing DNA primers and DNA polymerase. The temperature is raised until the DNA in the sample separates into single strands (at about 94^o - 96^o C), then lowered (50^o - 65^o 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 72^o 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.

Animation.

Process (video).

Products and protocols.

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 comparison using ancient bones (abstract).

DNA Sequencing and Genome Mapping

It was decided in the 1980's 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 wouldn't match your own sequence exactly.	Website of the Human Genome Project.
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 many important research organisms, and expanded to other 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.	History of sequencing. Many many genomes available.
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.	Drosophila. C. elegans (article). *Arabidopsis.*
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.	Application.

Genetic Engineering - Adding Genes

If you had a gene isolated that made an amazingly useful protein, wouldn't it be great if you could somehow just make the stuff? That was a question that arose as genes for important human proteins were isolated and sequenced. Transcription and translation, however, are not easily done in test tubes at a lab bench - living cells would need to be used. It was already known that bacteria can trade small gene packages, plasmids, that could sometimes become integrated into the main chromosome, putting a new gene into the genome. Procedures were developed to isolate plasmids, clip out their genes, and insert the target genes. Cultures would be exposed to the plasmids, but how to tell which cells integrated the genes? Marker genes were added to the plasmids. One common such gene produces a resistance to a particular antibiotic. After plasmid exposure and culture growth, the culture is dosed; cells that have not picked up and used the plasmids have no resistance and die.

CRISPR. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR Cas-9, C-associated protein 9. For gene editing within an existing genome, it is supposed to be cheaper, faster, more reliable, and more efficient than other methods. The system was adapted from a virus-defense system of bacteria – they clip out bits of viral DNA to use as targets if the viruses invade again. RNA binders are made with the viral DNA, and the Cas-9 comes in to expedite cutting up the targeted section of virus DNA. In the lab procedure, the RNA is from a targeted sequence in the target genome. Cas-9 helps to cut the DNA at that spot, and enzymes from the normal proofreading and repair systems can be used, clipping out pieces, and new alleles can be spliced in where “bad” alleles have been. Currently, the genes are in tissue cells, so the changes introduced are not passable to offspring. The changes could be introduced into embryos, though, and become part of a whole individual and inheritable.

Information on bacterial transformation.

Protocol description.

Troubleshooting problems.

Using the technique to produce human insulin.

Introduction to CRISPR.

CRISPR patent issues.

What's a Gene Really Do? - Knockout Mice.

Genome comparisons often highlight homologous genes in different species, but once you find a homologous gene, it isn't clear how the gene functions - does it do the same thing as the matching code? A way to determine this is to develop a strain of mice with that particular gene mutated into a non-working version. These are called knockout mice. A mutated sequence is developed and a line of embryonic stem cells are exposed to the code (often with a marker). Some of the cells will replace their allele with the mutated sequence. Those cells are implanted into early mouse embryos, in hopes that the altered cells will give rise to the reproductive system. Mutant heterozygous offspring are bred together, and the homozygous offspring will have two non-functional copies of the gene - what is the effect?

Making knockout mice.

Knockout mice available for research.

Lecture on knockout mice (video).

LINKS

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

General Biology 2 - Molecules and Cells

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