Fishing In A Molecular Sea

Date: January 18, 1999Labeling Kit Companies Labeling Kit Companies: Details In situ spatial localization of mRNA expressed only after ethylene treatment (left) compared to air grown seedlings used as a negative control (right). The RNA probe to the EIA0305 gene was labeled with fluorescein (NEL633) and visualized with NBT (Image courtesy of New England Nuclear) Remember the days when fishing for a sequence in the E. coli genome seemed like an overwhelming task? Somewhere along the way, it seems like the small pond we started fishing in has turned into an open sea. Today, with the entire human genome one of the hottest fishing spots around, the pool of genes waiting to be hooked seems ever increasing. As every seasoned angler knows, you need the right tools for the job--right rod, right line, right bait--and the same is true when you're fishing for gene sequences. If you're having trouble fishing out genes, maybe it's time to reevaluate your equipment! If your goal is to snag the only fish of its kind, you'll need a pretty sensitive probe. If you want to distinguish one species from another when they're all much the same, you'll need a highly specific probe or primer. And if you have a good catch that you need to analyze, you'll need a quick and easy method to do it. Is your nucleic acid probe best suited to your application? Are you wasting time labeling primers? Are you rooted in a lab tradition of labeling with a familiar kit? In this article, LabConsumer looks at the state of the art in nucleic acid labeling kits--from the tried and tested classic, nick translation, to innovative one-step chemical labeling--taking in novel features and modifications of new and old methodologies along the way. Both safety and financial concerns have fueled a trend toward nonradioactive labeling, and a number of kits are available for this purpose, many of which are based on methods originally used for radiolabeling. With a plethora of nonradioactive labels available for use and new labels emerging at an incredible pace, in depth consideration of individual labels is beyond the scope of this article. What we hope to bring you is an overview of current labeling methods so you can be sure you're using the right tool for the job and the sequence you're chasing doesn't go down in lab legend as "the one that got away." In determining the labeling method that's right for you, it's important to prioritize your criteria. It may be useful to consider the following list: Sensitivity--Is your target sequence found in low or high copy number? Specificity--Are you trying to distinguish a sequence from several similar sequences? Time--Do you label nucleic acids frequently? Do you work to a tight deadline? Would short incubation times increase your productivity? Convenience--Longer incubations may be most convenient for you, but do you have time to purify nucleic acid to be labeled, extract reaction components, or separate unincorporated label? Do you want to eliminate variability by using premixed reagents? Would you like to store kits at room temperature? Amount of starting material required--Are your labeling reactions limited by nucleic acid in short supply? Stability of probe--Will you need to use the same batch of probe in six months? Health and safety--Do you want to eliminate time-consuming monitoring, clean-up, and disposal regimens, while ensuring the safety of your staff? Cost--Do you want to reduce containment and disposal costs? Sensitivity is probably the primary concern of most researchers. For this reason, 32P is still the label of choice to ensure maximum sensitivity in detecting low-copy targets. Fluorescent labels--historically, the first real alternatives to radiolabeling--offer all the benefits of nonradioactive labeling but don't have the sensitivity needed in many applications. However, with the advent of chemiluminescent detection methods, nonradioactive labels started snapping at the heels of 32P. With an abundance of new labeling methods and novel labels to choose from, it's now impossible to ignore the options provided by this approach. RNA hybridization probes directed against mouse actin mRNA, prepared by T3 RNA polymerase-directed transcription using Molecular Probes' ChromaTide™ Cascade Blue®, BODIPY® FL-X, BODIPY TMR-X, Texas Red® and BODIPY TR-X UTPs. Unincorporated nucleotides are visible below the transcripts. Image provided by Molecular Probes. Safety and economics are primary advantages of nonradioactive labeling. Handling hazardous material is a surefire way to increase the stress level of any experiment, and disposal costs quickly mount up. In addition to nonradioactive labeling options that are available for most of the methods previously associated with radiolabels, novel methods are available, most of which are simple, often one-step procedures, and let's face it, any method is made easier when it doesn't involve contortions behind a protective screen! Depending on the method, one of the drawbacks of nonradioactive labeling is the amount of starting material required (sometimes micrograms rather than the nanograms required in, for example, random priming), which can be limiting for labeling materials in short supply. However, the stability of nonradioactive probes far exceeds that of their radioactive counterparts, so a single labeling reaction, with a hapten such as digoxigenin, can result in a uniformly labeled batch of probe that can be used for up to a year. Many seemingly novel labeling kits are available, based on old standards such as random priming, and many are suitable for both radioactive and nonradioactive labeling. Manufacturers have tweaked and polished classic kits, claiming improved sensitivity, often based on optimization of buffers and conditions. While these improvements are valuable for many applications, it's easy to fall into the trap of overinsurance. In considering your options, you may find that you're investing money in advanced technology you don't really need or that your money would be better spent on alternative methods. By virtue of its ability to label DNA to a high specific activity, Feinberg and Vogelstein's1 random priming method has long been a gold standard in labeling double-stranded DNA. The technique is based on priming DNA synthesis from random-primer-DNA complexes. Short random primers bind to complementary sequences of denatured DNA to serve as templates for the Klenow fragment of E. coli DNA polymerase I, which extends the primer sequences, incorporating labeled nucleotides as it does so. By eliminating the 5' -> 3' exonuclease activity of DNA polymerase I, use of the Klenow fragment ensures that labeled nucleotides are not removed from the newly synthesized duplex. Very small amounts of DNA can thus be labeled to a high specific activity to produce sensitive hybridization probes. Several manufacturers have incorporated novel variations into their random priming kits to improve labeling efficiency. Feinberg and Vogelstein originally recommended the use of random hexamers in the reaction, suggesting that binding of random sequences of this length would result in the most efficient and complete representation of target DNA in labeled probes. Since then, it has been demonstrated that use of longer random primers (nonamers or decamers) results in faster and more efficient incorporation of nucleotides. The increased stability of decamer-DNA complexes can increase efficiency of incorporation of labeled nucleotides by up to 80%. A band of interest from differential display was excised (panel A arrow), reamplified, and labelid with the HotPrime™ DNA Labeling Kit for Northern blot confirmation (panel B). Figure provided by GenHunter. GenHunter's HotPrime™ DNA Labeling Kit, in addition to using decamers, incorporates anchored oligo-dT primers to ensure cDNA probes are labeled throughout their length, with an eye toward probing differential display experiments. The Klenow fragment of DNA polymerase I retains the polymerization fidelity of the holoenzyme but does not degrade 5' termini because it does not have 5'->3' exonuclease activity. Several companies employ a Klenow fragment that has been genetically engineered to also remove the 3'->5' exonuclease activity of the standard Klenow fragment, dramatically increasing the stability of the probe and enhancing label incorporation in random priming reactions. Resulting increases in nonradioactive label incorporation allow nonradioactive probes to be used in some low-copy detection applications that were not previously possible using the Klenow fragment. TaKaRa Shuzo's Ladderman™ Labeling Kit takes advantage of a thermostable DNA polymerase isolated from Bacillus caldotenax YT-G (Bca DNA polymerase: TAK 2710B). Because the labeling reaction can be performed at a higher temperature (50-55°C), this polymerase allows stable polymerization through templates with high GC content (highly structured regions), unlike the E. coli Klenow fragment. The Prime-IT™ RmT kit from Stratagene also includes a thermostable polymerase, which enables the kit to be stored at room temperature. If reproducibility (or simply convenience) is a concern, Amersham Pharmacia Biotech offers Rediprime II, a kit in which all the reagents are premixed into a single, freeze-dried aliquot, reducing the risk of user error, and making them convenient for infrequent users or those with limited cold storage space. Roche Molecular Biochemicals/Boehringer Mannheim's High Prime Random Priming Labeling Kit or mix similarly provides a ready mixed random primed labeling solution, with a labeled nucleotide (DIG) already mixed in correct ratios for production of probes. If time is a determining factor in your choice of kits for random priming, reaction times range from 10 to 60 minutes (or overnight for very small amounts of DNA), so look for a kit at the low end of this range. Life Technologies' RTS RadPrime DNA Labeling system, for example, has predispensed single-use reactions that can prepare high specific activity probes in less than 10 min. Stripping Efficiency of StripAble™ vs. Standard RNA Probes. Duplicate Northern blots of mouse liver total RNA were hybridized with a 32P-labeled RNA probe complementary to mouse ß-actin synthesized using a standard transcription kit or Ambion's Strip-EZ™ RNA Kit. The StripAble RNA probe was stripped using the Strip-EZ Probe Degradation Buffer while the blot hybridized with the standard RNA probe was stripped with two washes of boiling 0.1% SDS. All blots were exposed overnight. Stripping probes in filter-based applications can have devastating consequences for the integrity of the filter or for the sensitivity of subsequent hybridizations and can be impossible in the case of RNA probes. To overcome this problem, Ambion has developed Strip EZ™ technology, which combines the convenience of random priming with an innovative probe-removal system. A modified dCTP, incorporated during probe synthesis, is easily degraded after hybridization and detection with a probe degradation buffer. Because the probe is cleaved into small fragments, it is easily removed by a mild wash. The Strip EZ system is also available for RNA and PCR probe preparation but is limited to labeling with gamma-32P dATP. Similarly, Roche Molecular Biochemicals' DIG High Prime and DIG DNA Labeling Kits include a labeled nucleotide, DIG-11-dUTP, that is alkali labile. The probe is degraded by a mild alkaline wash and easily stripped from membranes. In contrast to random priming, Ambion's Prime-A-Probe™ employs specific primers for the production of single-stranded DNA probes from PCR products or DNA fragments cloned into commonly used vectors. Prior to primer hybridization, the DNA vector is linearized distal to the sequence of interest so that run-off probe molecules are synthesized. A researcher's own primer/vector combination can also be used. The result is strand specific probes of a discrete length that cannot self-hybridize as do random-primed probes produced by priming from both strands of a double-stranded DNA template. Nick translation, where it all began, has been displaced somewhat by the high specificity probes offered by random priming, but some novel variations in nick translation kits ensure that this method remains a player. The method relies on the 5'->3' polymerase activity of DNA polymerase I, coupled with the enzyme's 5'->3' exonuclease activity. To initiate the reaction, random nicks are created in double-stranded DNA by DNase I. Nucleotides are added sequentially to the 3' end of the nick via the 5'->3' polymerase activity of DNA polymerase I, while the enzyme's 5'->3' exonuclease activity serves to remove nucleotides from the adjacent 5' terminus to make way for the newly synthesized strand. The existing nucleotide sequence is thus renewed, with the inclusion of labeled nucleotides, but no net synthesis occurs. Incorporation of nucleotides rarely exceeds 40% to 60%, and specific activities are commonly 10 times lower than those obtained with random priming. Not too many surprises are available in the kits, and some companies have discontinued their products because of low demand. Some of the kits are optimized for use with proprietary labels, such as Vysis fluorophore-labeled dUTPs. Vysis's Nick Translation Kit is designed for labeling DNA for use in fluorescence in situ hybridization. In CLONTECH's Biotin-21-dUTP Nick Translation Kit, the biotin-21-dUTP (where 21 denotes the number of atoms in the spacer arm) has the longest commercially available spacer arm. The general rule of thumb is the longer the spacer arm, the lower the efficiency of label incorporation. However, by reducing steric hindrance, longer spacer arms increase accessibility of labels to detection reagents, thus increasing the sensitivity of detection. Using biotin-21-dUTP coupled with chemiluminescent detection, probe sensitivity can be as low as 1 pg of nucleic acid. Of course, increasing sensitivity can also increase background, so choose your spacer arm with care. Protruding, recessed, or blunt-ended double- or single-stranded DNA molecules can be labeled in a reaction catalyzed by terminal transferase. In the presence of Co2+ ions, terminal transferase, a template-independent polymerase used in most 3'-end labeling kits, catalyzes the addition of labeled nucleotides to the 3' hydroxyl terminus of the template. By using a labeled dideoxynucleotide, a single labeled nucleotide can be added to the end of the DNA molecule. Additional labeled nucleotides can be added, increasing the sensitivity of the probe, if the dideoxynucleotide is replaced with a labeled deoxynucleotide. A variety of labeling kits is available for the addition of single or multiple labels (both radioactive and nonradioactive) to DNA or oligonucleotides. With Roche Molecular Biochemical's DIG Oligo 3' End Labeling Kit, terminal transferase is used to add a single DIG-11-ddUTP to the 3' end of an oligo. In its 3' Oligo Tailing Kit, Roche Molecular Biochemicals has replaced the DIG-11-ddUTP with DIG-dUTP and dATP at a ratio of 1:10, resulting in the addition of a 50-nucleotide tail on average. The dATP serves to space out the DIG-dUTP to increase the efficiency of incorporation and antibody recognition in the detection reaction. The increased incorporation of DIG label increases sensitivity, but the added dATP can lead to nonspecific binding. If specificity is your primary concern, the 3' End Labeling Kit is the kit to choose. Amersham Pharmacia Biotech's 3' end labeling kits are also available with either ddUTP or dUTP. The ECL 3' Oligolabeling System can be used for labeling with fluorescein-11-dUTP, resulting in a tail of six to eight nucleotides, promising good sensitivity and low background. The ECF 3' Oligo Labeling System adds a fluorescent tail to oligos, which can be detected using fluorescence scanning instrumentation. Enzo Diagnostics offers 3'-oligonucleotide end labeling and tailing kits that include dNTPs or ddNTPs labeled with nonradioactive labels attached via spacer arms of different lengths, such as biotin-16-dUTP or biotin-7-dATP. These options provide flexibility in terms of sensitivity and overcoming background signal. To label the 5' end of DNA, RNA, or oligonucleotides for use in sequencing, PCR, nuclease protection assays, or filter-based hybridizations, T4 polynucleotide kinase can be used to catalyze the transfer of the gamma-phosphate of a ribonucleoside 5'-triphosphate donor such as gamma-32P ATP to the 5' hydroxyl group of the nucleic acid to be labeled. For 5' labeling of most nucleic acids, a 5'-phosphate must first be cleaved from the 5' end by alkaline phosphatase, leaving a 5' hydroxyl group that can accept the gamma-phosphate. Phenol extraction and ethanol precipitation are then required before phosphorylation can be completed. Amersham Pharmacia Biotech's 5' End Labeling Kit, Ambion's KinaseMax™ Kit, or TaKara Shuzo's MegaLabel™ DNA Labeling Kit offer valuable time-saving alternatives by eliminating the need for the alkaline phosphatase step and the subsequent extractions to remove this enzyme. In Amersham's system, the phosphorylation reaction is driven by excess ADP, which causes the enzyme to transfer the terminal 5' phosphate from DNA to ADP. The DNA is then rephosphorylated by transfer of the labeled gamma-phosphate from gamma-32P ATP, resulting in specific activities in excess of 1 x 106 cpm/pmol. In addition to enzyme-mediated labeling reactions, kits are available for chemical labeling of nucleic acids, primarily with nonradioactive labels. Enzymatic labeling can yield a variety of products with different annealing properties, which can make characterization difficult in some cases. In addition to ease of labeling (some kits are based on simple one-step reactions), the beauty of chemical labeling is that the initial substrate remains intact after labeling, allowing quantitative recovery of labeled products. Chemical labeling kits rely on a number of different technologies for attaching label to the nucleic acid molecule. Like ethidium bromide, psoralens can be incorporated into the nucleic acid structure. Both Ambion and Schleicher and Schuell offer a psoralen-biotin labeling system (the BrightStar Psoralen™ -Biotin Nonisotopic Labeling Kit and the Rad-Free Labeling System, respectively). Psoralens are planar tricyclic compounds that intercalate between bases, becoming covalently attached to the nucleic acid upon irradiation with long-wave UV light. Psoralens preferentially intercalate between thymidine in DNA and uridine in RNA so they can be used to label any type of nucleic acid. Different types of nucleic acids can be labeled simultaneously, thus eliminating any discrepancies in labeling efficiencies when comparing multiple probes. Advantages of labeling with psoralen-biotin include the following: the probe is stable for at least a year when stored at -80°C; the reaction is not dependent on the amount or purity of the template, as are enzymatic labeling methods; and sensitivities can be comparable with those of 32P radiolabeled probes (100 fg), depending on the biotin detection method used. An appropriate long- wave UV lamp (medium- and short-wavelength lamps are not suitable) is required, and some of the labeling steps must be conducted in low light, seemingly small prices to pay for the many advantages of this system. Although psoralens intercalate into the nucleic acid structure, they shouldn't be confused with ethidium bromide when considering safety issues. Intercalation occurs only under UV light, so the reagents are not biohazardous in normal benchtop conditions. In contrast to proteins, nucleic acids have few reactive groups that are accessible for modification, limiting options for chemical labeling. One approach to overcome this problem is to attach reactive linkers, such as thiols or amines to the nucleic acid, opening up options for labeling with the many available maleimide-containing or succinimydyl ester forms of dyes and labels that are otherwise limited to protein labeling. Diagram of the FastTag™ reaction from Vector Laboratories Vector Laboratories offers a novel approach to labeling via a universal linker, to which any sulfhydryl-reactive compound can be attached. Using the FastTag™ system, haptens, fluorochromes, affinity ligands, or other markers can be coupled to any nucleic acid-DNA, RNA, or oligonucleotides. The disulfide-containing universal linker, FastTag reagent, is conjugated to the target nucleic acid by heat or light (using a thermal block cycler or exposure to light). After the disulfide bond in the FastTag linker arm is reduced to yield a free thiol, a covalent bond can then be formed between the FastTag reagent thiol and a maleimide-containing hapten, fluorochrome, sugar, affinity ligand, or other marker. The system is enzyme free, economical (up to 100 times more nucleic acid can be labeled with one FastTag kit than with similarly priced enzyme kits), and versatile. Labeling takes less than 1 hour, and sensitivity can be in the subpicogram range for some applications, comparable to that achieved using 32P-labeled probes. The method of choice for labeling oligonucleotides that will be subject to enzymatic action at the 3' end, such as primers for sequencing or PCR, is 5' end labeling. While enzymatic labeling at the 5' end is possible with polynucleotide kinase, it is limited to labeling with 32P. However, chemical attachment of nonradioactive labels at the 5' end is an alternative, via a 5' amino linker. Many amine-reactive reagents such as succinimydyl esters are available for labeling protein molecules, but the nonbasic "amines" of adenosine or guanosine are virtually unreactive with these agents. To label oligonucleotides directly with these reagents, amines must be incorporated during synthesis of the oligonucleotide. Roche Molecular Biochemicals' DIG Oligo 5' End Labeling Kit includes reagents required to create a 5' terminal amino function, created during a final synthesis step when the oligonucleotide is reacted with an amino-linker phosphoramidite, a modified building block used in oligonucleotide synthesis. After synthesis, a succinimydyl ester of digoxigenin binds to the oligonucleotide via the amino linker. Because this kit includes the amino-linker phosphoramidites, it is best suited to core facilities where oligonucleotides are synthesized. Roche Molecular Biochemicals' Alkaline Phosphatase (AP) Oligo Labeling Kit works on a similar principle to the DIG Oligo 5' End Labeling Kit--an oligo with a 5' amino terminal function is activated with an activation reagent, and AP is then conjugated directly--but the amino-linker phosphoramidite is not included, so it is suitable for use only with oligonucleotides containing a 5' terminal amino group. Perhaps the widest range of kits for labeling oligos synthesized with a 5' terminal amino group is available from Molecular Probes. FluoReporter Oligonucleotide Amine Labeling Kits are available with a variety of amine-reactive succinimydyl esters--including biotin, BODIPY, DNP, carboxytetramethylrhodamine (TAMRA), and fluorescein, to name but a few--most of which contain aminohexanoic spacers to reduce the label's interaction with the oligonucleotide and enhance its accessibility to secondary detection reagents. FluoReporter Oligonucleotide Phosphate Labeling Kits permit the single-step covalent labeling of 3'- or 5'-phosphate-terminated oligonucleotides. These kits can also be used to double-label oligonucleotides radioactively labeled at the 3' end or, in combination with T4 polynucleotide kinase, to fluorescently label oligonucleotides lacking a 5'-phosphate. The phosphate labeling kits can also be used to label linear DNA restriction fragments. For direct covalent labeling, six Label IT™ Nucleic Acid Labeling Kits are available from the Mirus Corporation (distributed by PanVera) for labeling with rhodamine, fluorescein, digoxin, biotin, Cy3, and Cy5. Using proprietary technology, the kits offer one-step reproducible labeling that can exceed one label per every 10 nucleotides. The kits work equally well with DNA (double stranded, single stranded, supercoiled, or linear) or RNA, and leaves the nucleic acid intact. Labeled DNA can be detected at the sub-picogram level by chemiluminescent detection, making it useful for a range of applications, including intracellular localization studies. The ECL Direct Kit from Amersham Pharmacia Biotech can be used for direct labeling of DNA or RNA with horseradish peroxidase (HRP). The protocol takes only 20 minutes and the DNA need not be purified. The concept is based on electrostatic binding of the positively charged HRP complex with negatively charged denatured or single-stranded DNA or RNA. Glutaraldehyde serves to covalently cross-link the HRP to the nucleic acid. Amersham Pharmacia Biotech advertises ECL Direct as its fastest and easiest system for high target applications such as colony or plaque screens or genome mapping. Detection of HRP activity is by chemiluminescence via HRP-catalyzed oxidation of the substrate Luminol. Using the same principle, the AlkPhos Direct Labeling Kit is used to label DNA with a thermostable AP, allowing the control of stringency with salt or temperature and the speed of a direct labeling system. Roche Molecular Biochemicals' Biotin or Digoxigenin Chem-Link Labeling Kits rely on noncovalent binding of a cis-platinum reagent (Chem-Link) to the N7 position of guanosine and adenosine bases, which can result in binding of a biotin or digoxigenin label every 10 bases. The kits are suitable for labeling double- or single-stranded DNA, but the reaction occurs at 85°C, so it's not suitable for short oligonucleotides that may be unstable at this temperature. The amount of material required for labeling,1-2 µg, is on the high side (compared to other methods such as random priming), but it need not be purified from low-melt agarose. An advantage of Chem-Link is that is allows researchers to screen large pools of RNA. Instead of using cDNA as a probe for differential display experiments, researchers can label an RNA pool directly with Chem-Link , skip the cDNA synthesis and then use the labeled RNA as a probe, increasing specificity and sensitivity. Available kits for labeling oligos via a 5' amino linker offer the flexibility of labeling with a choice of aminoreactive reagents, but if you have a specific label in mind, oligonucleotidess can be modified during synthesis to incorporate labeled phosphoramidites. CLONTECH's Easy-ON phosphoramidites are used exactly like normal DNA phosphoramidites and directly incorporate labels at the 5' end via an aminohexanol spacer. CLONTECH's Label-ON™ reagents can be used for direct incorporation of single or multiple labels, at the 5' or 3' ends (3' labeling is achieved by using Label-ON CPG solid supports), or internally, during oligonucleotide synthesis. The Label-ON reagents have a proprietary 2-aminobutyl-1,3-propanediol (ABPD) backbone that confers specific labeling advantages. Multiple labels can be incorporated-in fact, different labels can be incorporated into the same oligonucleotide to produce a multifunctional probe. When incorporated internally, natural internucleotide distance is maintained. The ABPD backbone has a seven-atom spacer arm that separates the attached label from the phosphodiester connection point for reduced steric hindrance, resulting in improved hybridization and minimized duplex destabilization. In addition to postsynthesis labeling of RNA, RNA probes can be prepared directly by in vitro transcription. High specific activity RNA probes for use in nuclease protection assays, in situ hybridizations, and filter-based hybridizations such as Northern and Southern blotting can be synthesized by transcription of an insert cloned into an appropriate vector. Transcription can be from SP6, T7, or T3 phage polymerase promoter sequences on the cloning vector (kits may include one, two, or all of these polymerases) and incorporates modified nucleotides. On paper, there's not difference between the kits--most manufacturers claim their kits are optimized for the efficient synthesis of full-length probes with specific activities in the region of 109 cpm/µg. Epicentre Technologies claims that its AmpliScribe™ T7, SP6, and T3 High Yield Transcription Kits can yield up to 30 times more RNA per reaction than conventional transcription reactions, while Ambion touts the specificity of the highly purified polymerases found in its kits. Enzo Diagnostics offers transcription kits with flexibility in the choice of modified ribonucleotides--fluorescein, biotin and digoxigenin--as well as a kit, BioArray™, specifically designed for use with nucleic acid arrays. To overcome low specific activity probes, NEN offers an amplification system, Tyramide Signal Amplification Kit that can enhance non-radioactive signals more than 100-fold in situ. With so many kits to choose from, covering a wide range of labeling options, it may seem like fishing for the best kit is as hard as fishing for your target sequence. Now that you've seen what's available, it may be helpful to reconsider the checklist at the beginning of this article. You may find that you can tailor your nucleic acid labeling to save time, money, headaches, and increase both productivity and your chances of fishing out your target sequence. Labeling Kit Companies Labeling Kit Companies: Details A.P. Feinberg and B. Vogelstein, "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity," Analytical Biochemistry, 132:6-13, 1983. The author can be contacted at paladichuk@home.com.

Fishing In A Molecular Sea

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