Molecular Biology Reagent Kits Simplify Lab Procedures

Companies discussed in the accompanying article: Bio-Rad Laboratories 2000 Alfred Nobel Dr. Hercules, Calif. 94547 (800) 4 - BIORAD Fax: (800) 879-2289 Products: Call for catalog BIO 101 Inc. 1060 Joshua Way Vista, Calif. 92083 (800) 424-6101 Fax: (619) 598-0116 Products: phage DNA isolation yeast transformation Geneclean DNA purification Mermaid oligomer purification RNaid RNA purification Circleprep plasmid preparation G nome DNA isolation, for genomic DNA BioWhittaker Inc. 8830 Biggs F

By | February 8, 1993

Companies discussed in the accompanying article:

Bio-Rad Laboratories
2000 Alfred Nobel Dr.
Hercules, Calif. 94547
(800) 4 - BIORAD
Fax: (800) 879-2289
Products: Call for catalog

BIO 101 Inc.
1060 Joshua Way
Vista, Calif. 92083
(800) 424-6101
Fax: (619) 598-0116
phage DNA isolation
yeast transformation
Geneclean DNA purification
Mermaid oligomer purification
RNaid RNA purification
Circleprep plasmid preparation
G nome DNA isolation, for genomic DNA

BioWhittaker Inc.
8830 Biggs Ford Rd.
Walkersville, Md. 21793-0127
(800) 638-8174
Fax: (301) 845-8291
Limulus Amebocyte Lysate
Gel-Clot LAL
Chromogenic LAL

Epicentre Technologies
1202 Ann St.
Madison, Wis. 53713
(800) 284-8474
Fax: (608) 251-3199
Ampligase thermostable DNA ligase
SequiTherm cycle sequencing
AmpliScribe transcription

Oncor Inc.
209 Perry Pkwy.
Gaithersburg, Md. 20877
(800) 77-ONCOR
Fax: (301) 926-6129
Oncor probes
Chromosome in situ hybridization systems
Probe tech systems for DNA analysis
RNA/DNA in situ reagent

Flip through the pages of the major science journals, and you'll find ads for kits that enable a researcher to do nearly anything imaginable--detection or purification, for example--to nucleic acids, chromosomes, and proteins. Such ads are starting to trickle into the medical journals, also, as physicians begin to replace or supplement traditional microbiology-based tests with DNA probes or monoclonal antibody kits to diagnose infections.

With labs in the biotechnology industry and the universities busier than ever and researchers in the Human Genome Project and here generating a constant stream of new material for kit development, the future market for molecular biology kits looks substantial.

A reagent kit, also often called a "system," consists of the chemicals needed to perform a given laboratory procedure, packaged with directions on their use. The catalog from Bio-Rad Laboratories in Richmond, Calif., provides a somewhat sweeping definition of such kits as "systems that enable scientists to recognize and decipher the multiple chemical patterns which are the clues to understanding life, human health, and materials science."

What's In A Reagent Kit? "A reagent kit is an assembly of all components, optimized and pretested, so they give good results without the user needing to do a lot of work beforehand," says Gary A. Dahl, president of Epicentre Technologies of Madison, Wis.

"The idea is to produce something that will help researchers do the simple things so they can get on with the experiments, which is what they are supposed to be doing," adds Jim Gautsch, vice president of kit development at BIO 101 Inc. of Vista, Calif.

Most kit producers also offer flexibility.

"All of our products are sold in kit forms, and you can buy the reagents separately, but that is more expensive," says Annette Short, customer service representative at Gaithersburg, Md.-based Oncor Inc. Oncor calls itself a "molecular pathology" company specializing in using DNA probes to highlight chromosome abnormalities behind inherited disorders and cancer. "We try to offer laboratories the ability to customize particular needs, not forcing them into a kit," Short says.

In molecular biology, the number of procedures actually covered by the many reagent kits available is not as daunting as the bulging catalogs might suggest.

Despite the large number of molecular reagent kits on the market, the procedures they facilitate--and the molecules they target-- are comparatively few. The job of choosing an appropriate kit is simplified by first identifying the task to be performed and then matching it with the molecule involved.

The most common molecules, of course, are DNA, RNA, and proteins. And the most common procedures are detection, extraction, isolation, purification, synthesis, sequencing, labeling, modification, or mutagenizing.

A typical reagent kit includes enzymes, controls, buffers, labeling reagents, possibly DNA probes or antibodies, and the all-important protocol. Very helpful are the newsletters published by reagent kit manufacturers, in which users describe applications of the products. For example, Cleveland-based U.S. Biochemical Corp.'s "Editorial Comments," Oncor's "The Molecular Cytogenetics Newsletter," and La Jolla, Calif.-based Strategene Corp.'s "Strategies in Molecular Biology" provide helpful hints and new applications.

The challenge in the reagent kit business is to stay at the cutting edge of research and be among the first to recognize when a new approach both fills a niche and can save a scientist time if its components are prepackaged together. One way that a company can do this is to tap into the experiences of its staff, says BIO 101's Gautsch.

"I came from the Scripps Clinic [of La Jolla, Calif.], where I spent a lot of time in the lab making basic materials so I could go on [with research]," Gautsch says. "Our kits allow you to do procedures that [otherwise would] slow you down, that often are stumbling blocks."

He cites as an example Geneclean, a kit that removes impurities and inhibitors (such as organic solvents, unreacted nucleotides, and salts and proteins) from agarose gels, ensuring that results on subsequent experiments are reliable.

Kit Genesis New kit ideas can come from a variety of sources. "We have a scientific advisory board of people in the field either working on projects or keeping their eyes open to what's coming up," says David Bruning, genetics marketing manager at Oncor. "We also have scientists in house who scour papers [in the literature]. We get feedback from going to meetings, information from customers, and from customer service reps."

Many considerations enter into deciding which new DNA probes to commercialize as a kit. Would research labs actually use a new probe kit, or are they already using their own versions? How many people have a disorder caused by the gene detectable with a new DNA probe? Would physicians ultimately use the new DNA probe kit to diagnose the associated illness, or do diagnostic kits using other technologies already exist? Are other companies close to developing a kit using the particular probe?

Knowledge about the disease in question is important, too.

"Sometimes a disease is not well enough characterized to judge if a specific probe is useful," says Bruning. He cites, for example, the phenomenon of genetic heterogeneity, in which a set of symptoms is caused by more than one gene. A diagnostic test based on a DNA probe to one gene will yield a false negative result for patients whose problem is caused by another gene.

Assembling a kit that not many others have thought of is one approach at Epicentre Technologies. This firm set its sights on kits based on the ligase chain reaction (LCR, also known as ligation amplification). LCR is an alternative gene amplification technology based on the ability of ligase to knit together pieces of a gene. It has diagnostic uses--if a gene from an infectious microbe or virus is present in a patient's sample, the ligase will join the pieces, which are then detected.

"Our Ampligase kit contains everything necessary for ligation amplification, with the customer's own target-specific set of four oligo-deoxynucleotides," says Dahl. The kit includes buffers, ligase, and a positive control template.

Another strategy in reagent kit development is to troubleshoot, identifying a problem and solving it. Dahl describes a problem in sequencing DNA that is avoided with Epicentre Technologies' Sequi-Therm cycle sequencing kit.

"DNA fragments with regions rich in the DNA bases C and G tend to be lost, so if you read down the four lanes of a [sequencing] gel, you miss certain areas of the sequence," he says. This is more than just a minor annoyance, because GC-rich areas are often present very near to genes that encode protein, and are also the parts of genes that most frequently mutate, causing disease.

Carving A Niche Oncor Inc. is focusing on a fusion of technologies--DNA probes with cytogenetics (identifying chromosomal variants that cause illness). This technology is called "FISH," for fluorescence in situ hybridization, or chromosome painting, and was pioneered at Lawrence Livermore National Laboratory, Livermore, Calif.

In FISH, a labeled piece of DNA, the probe, binds to its complementary sequence on a chromosome. This is much more specific than classical chromosome staining, in which dyes are used to stain chromosomes in characteristic patterns reflecting more generalized features, such as GC- vs. AT-rich areas.

FISH is very useful in distinguishing among some blood cell cancers, which are often associated with chromosomal rearrangements. A definitive diagnosis is crucial to determining prognosis and course of treatment. This diagnosis is possible by deciphering the associated chromosomal anomaly.

A classic example is chronic myelogenous leukemia (CML), which is nearly always associated with a chromosome translocation that places the tip of one chromosome, number 9, onto chromosome 22 (chromosomes are numbered in size order). This so-called Philadelphia chromosome, named for the city where it was discovered in 1960, juxtaposes the Abelson (or ABL) oncogene on chromosome 9 against a region of chromosome 22 called the breakpoint cluster region, or BCR. When the two genes are opposed, a single "fusion" protein is manufactured under their direction. This protein somehow lifts cell cycle control to cause cancer.

Oncor's BCR/ABL Translocation DNA Probe highlights the ABL gene with yellow-green fluorescein and BCR with red rhodamine. If a patient with unexplained fatigue and bruising and a leukemic white blood cell count has the red and green flash next to one another in a chromosome preparation, the telltale Philadelphia chromosome is there, and the diagnosis is CML.

"We also have a Translocation 15/17 Probe for acute promyelocytic leukemia, neuroblastoma, and her 2 neu, which is an oncogene associated with breast cancer," says Bruning.

Oncor's Chromosome In Situ System for Dual Color Detection includes reagents to block repetitive DNA so that the probes home in on the single copy genes of interest; reagents for hybridization, detection, and signal amplification; and the probe. The dual color technology is licensed from Indianapolis- based Boehringer Mannheim Corp.

Perhaps most exciting is the use of Oncor's probe kits to solve chromosomal mysteries. Vicki E. Powers and Huntington F. Willard of the department of genetics at Stanford University used the probe kits to identify isochromosomes more definitively than is possible with traditional staining. Isochromosomes have identical arms because during cell division, the duplicated chromosome split along the wrong plane. The result is double doses of some genes and the absence of others--an unhealthy imbalance of genetic material. "The information obtained [from using the probes] both aids diagnosis and provides fundamental information on the nature of human chromosome abnormalities," they reported in the company's newsletter.

Another chromosomal mystery is being solved by David F. Callen of the department of cytogenetics and molecular genetics at Adelaide Children's Hospital in Australia. He is using Oncor's DNA probe kits to determine the chromosomal origins of ring chromosomes. These tiny rings of genetic material are sometimes present in cells of patients with unrecognized syndromes. Being able to tell which chromosome gave rise to the ring can aid diagnosis.

While Oncor reaches both the research and clinical marketplace, other firms specialize in molecular biology reagent kits for the health care industry. Here, too, innovation, convenience, and time-saving are the goals.

For example, there is the PACE 2-Direct Probe Assay for Chlamydia trachomatis and Neisseria gonorrhoeae, offered by San Diego-based Gen-Probe. A dual test is useful because a patient who has one of these sexually transmitted diseases has up to a 50 percent chance of having the other. The kit includes the probes, an activator, a separation reagent, probe diluent, positive control, negative reference, and a wash solution, sufficient for 100 specimens. A single test uses one urogenital swab and replaces standard microbiology culturing. Such kits serve a need because physicians do not have the time or training to develop DNA probes.

A Crustacean Simulation The quintessential molecular biology kit is one that provides a test that cannot be done--at least not easily--otherwise. This is so for the Limulus amebocyte lysate (LAL) test, a standard method for detecting bacterial endotoxin in drugs or on medical devices such as syringes. Endotoxin causes septic shock, which can result in death.

The LAL test borrows the distinctive response of the horseshoe crab (Limulus) to encountering endotoxin--its coppery blue blood gels. This reaction was discovered in 1956 by Frederick Bang at the Marine Biological Laboratory at Woods Hole, who, with colleague Jack Levin, isolated the amebocytes responsible for the reaction, and re-created the clotting in vitro. The LAL test rapidly replaced injecting rabbits with contaminated drugs and looking for a response of fever, indicating the presence of endotoxin.

Today, companies that market the LAL test usually get freeze- dried crab blood from Associates of Cape Cod Inc., Falmouth, Mass. Here, during summers, college students "bleed" horseshoe crabs that hang upside down, the animals' blood collecting in jugs below them. (Only 30 percent of each crab's blood is taken, and the crabs are returned, apparently unscathed, to the sea.)

Because most manufacturers of medical devices can't simply pluck a horseshoe crab from a nearby shore and watch its blood clot into blue gelatin, companies such as Bio-Whittaker Inc. of Walkersville, Md., provide LAL kits.

"Our product is derived from the amebocyte," says product manager Maribeth Donovan. "We lyse the cell, and all the components of the blood coagulation system are there. The LAL test consists of lysates of the blood cells. You mix a sample with LAL, and if it gels, it means endotoxin is in the sample."

More informative is the company's "chromogenic" version of the LAL test.

"Instead of the flipped tube having solid on top, it turns yellow," says Donovan. "We can quantitate the amount of endotoxin in the sample by the intensity of the yellow compared to a standard curve."

Ricki Lewis is a freelance science writer based in Scotia, N.Y. She is the author of a biology textbook and has just completed a human genetics text.

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