Lumigen, Inc. - Chemistry that brings meaning to light

Principles

Replication of nucleic acid templates by the primer-assisted Ligation of Multiple Oligomers (LMO) represents a new technology pioneered at Lumigen. LMO involves the simultaneous enzymatic ligation of a set of contiguous oligomer 5'-phosphates with one or more primers on a complementary template. Primer/oligomer size and reaction conditions are designed such that primers stably hybridize but oligomers of about 5-10 nucleotides do not. Ligase initiates a cascade of consecutive ligations with a high degree of specificity only when a primer is hybridized. All subsequent ligations are prevented as well.

LMO makes it simple to specify the length of a strand being synthesized in advance. The length is specified by the combined length of the primer(s) and the set of consecutive oligonucleotides. The requirement of contiguous alignment of primers and short oligomers is absolute. Gaps in between adjacent segments of even one nucleotide terminate the process.

The endpoints of the reaction product strand are precisely controllable by simply supplying only the desired oligomers. Ladders of differing molecular weights separated by known increments are readily prepared. Constant size increments shown oligomers, e.g. octamers.

Ligation can also be terminated by the use of an oligomer which can not support further enzymatic ligation. Oligomers having a dideoxy base at the 3' end can not be ligated to the subsequent oligomer and block further ligation.

A unique feature of LMO which sets it apart from polymerase-based extension methods is the ability to extend a primer from either end. A series of short oligomer 5'-phosphates can be ligated off the 3' end of a primer or, provided the primer has a 5'-phosphate, off the 5' end of the primer or off both ends simultaneously by bidirectional ligation.

Templates

• Template may be of any size from short synthetic polynucleotides of 50-100 bases to restriction fragments up to genomic DNA
• RNA or DNA templates can be used

Types of ligase

• Both ATP-dependent and NAD-dependent ligase enzymes can be employed in LMO applications
• ATP-dependent ligase enzymes such as T4 DNA ligase will accept oligomers as short as pentamers as substrates for ligation
• NAD-dependent ligase enzymes such as Ampligase (Epicentre Technologies) are suitable for higher temperature reactions but require somewhat longer oligomers

The three images above show the relative ability of different ligase enzymes to join a small oligonucleotide-5'-phosphate to the 3' end of a primer oligonucleotide hybridized to a CFTR gene template. Primer oligonucleotide was ligated (from left to right) with one biotinylated oligomer in each reaction having 6, 7, 8, 9, 10 or 11 bases. Ampligase and Taq ligations proceeded at 45°C for 1 h, the T4 ligation for Southern blot.
 

LMO Advantages

The LMO technology offers several advantages and unique capabilities. Being a broad based methodology applications range from nucleic acid replication, labeling and amplification to sequence-specific detection, mutation analysis and gene expression profiling. No other nucleic acid replication technology enables all of the following features.

Flexibility in choosing direction of extension:

• Allows mutations to be differentiated or ignored
• Highly repetitive regions can be avoided
• Unidirectional or bidirectional
• RNA or DNA template

Replication length controllable by choice of oligomer set:

• Extension stops when oligomer is excluded
• Precise control of labeling
• Density of label incorporation by design
• Different types of labels - chemiluminescent, fluorescent, enzymes, branched multi-labeled arms, etc.
• Multiple labels on an oligomer

Unique nucleic acid amplification capabilities:

• Selective amplification of desired DNA or RNA sequence by selection of oligomers; i.e. can be mutation specific
• Multiple primers can be used on either or both strands
• Can use labeled oligomers to make amplified labeled product

 

Applications

Most techniques which involve a polymerase extension or a ligation reaction for nucleic acid synthesis can be adapted to LMO. The unique features of the technology provide new ways to accomplish established techniques including:

Probe Labeling

• Regular controlled density labeling of ss or ds DNA
• Can accommodate many different types of labels
• Hapten labels allow immunological detection or solid phase capture

Amplification

• Linear or exponential amplification by thermocycling protocol using a thermostable ligase enzyme such as Ampligase
• Amplification factors of greater than 1 million-fold
• Allele specific amplification
• Amplicon can have blunt ends or overhangs

Mutation Analysis

• Primer-assisted ligation of labeled oligomers can be used in diagnostic methods for detecting point mutations
• A variety of formats can be developed
• A simple yes/no test can be devised with sets of oligomers for the wild type and mutant sequences where one set carries a detectable label
• Genotypes can be distinguished using two sets of oligomers with the wild type and mutant sequence oligomers carrying different labels
• Different mutations in a gene can be differentiated with mutation-specific sets of differently labeled oligomers

Infectious Agent Detection

• LMO can be used to develop diagnostic methods for detecting bacterial or viral infectious disease-causing agents
• The ability to controllably label nucleic acids to high density by ligation of oligomers improves detection sensitivity
• Large analyte-specific probes need not be prepared in advance.

Gene Expression Analysis

 

Resources

The core technology underlying LMO is described in four U.S. patents issued to Lumigen. Additional patent applications are pending worldwide.

Issued U.S. Patents Relating to LMO

• U.S. 6,020,138 - Methods for detecting nucleic acids by ligation of multiple oligomers, Issued February 1, 2000
• U.S. 6,013,456 - Methods of sequencing polynucleotides by ligation of multiple oligomers, Issued January 11, 2000
• U.S. 6,001,614 - Novel Methods of synthesizing labeled polynucleotides by ligation of multiple oligomers, Issued December 14, 1999
• U.S. 5,998,175 - Methods of synthesizing and amplifying polynucleotides by ligation of multiple oligomers, Issued December 7, 1999

LMO Poster Presentations

• Posters at the 33rd Oak Ridge Conference, Seattle, WA, May 4-5, 2001
• Nucleic acid amplification through ligation of multiple oligomers (LMO). Akhavan-Tafti H, Handley RS, Reddy LV, Sugioka Y, Siripurapu S, Schaap AP, Schlederer T, Raberger B, Bartonek K.
• Applications of multiple oligonucleotide ligation-based amplification. Akhavan-Tafti H, Handley RS, Reddy LV, Sugioka Y, Siripurapu S, Schaap AP, Schlederer T, Raberger B, Bartonek K.
• H. Akhavan-Tafti, R. DeSilva, K. Sugioka, R. Handley, A. P. Schaap, "Acridan Phosphates as Chemiluminescent Labeling Compounds." Lecture at the IXth ISLS 2000 Luminescence Symposium in Montpellier, France, May 15-17, 2000.
   

LMO Technology - Labeling & Detection

LMO strand replication can utilize labeled primers as many other techniques do, but the real power of LMO rests in the ability to build a nucleic acid strand with labeled short oligomers. Other known methods of preparing labeled nucleic acids such as end tailing, nick translation and random prime labeling do not afford control of the spacing or quantity of label incorporation.

When LMO is used for preparing a labeled oligonucleotide or polynucleotide:

• The density, spacing and nature of labels can be precisely specified
• Any type of label can potentially be incorporated, including branches, linkers, enzymes
• Precisely controlled multiple labels and mixtures of labels can be used to design energy transfer assays
• One or both strands can be uncontrollably labeled

The ability to exactly control label incorporation is illustrated in a set of LMO reactions in which sets of contiguous octamers with alternate members carrying a single hapten label were ligated onto a primer-template hybrid. The experimental design is depicted in the scheme above. The resulting reaction mixtures were separated by SDS-PAGE, blotted and visualized with an enzyme-labeled anti-hapten antibody and a chemiluminescent enzyme substrate. The pattern of bands observed reflects both the increasing size and number of labels in the products.


Lane 1 - 3 octamers, 1 label
Lane 2 - 4 octamers, 2 labels
Lane 3 - 5 octamers, 2 labels
Lane 4 - 5 octamers, 3 labels

Precise labeling can aid in improving precision in quantitative assay applications. High density labeling can be achieved by labeling each short oligomer. Densities as high as one label per every five bases can be achieved by using labeled pentamers. Improvements in detection sensitivity can be expected with greater label incorporation.

Label Types

LMO strand replication is fully compatible with the use not only of labeled primers but also with labeled short oligomers. Given the wide variety of methodologies for labeling oligonucleotides LMO offers almost unlimited potential for incorporating labels into product nucleic acid strands.

Some kinds of labels which can be incorporated into LMO protocols include:

• Directly detectable labels including chemiluminescent, fluorescent, chromogenic, and radioisotopic labels
• Indirectly detectable labels, for example, small haptens such as biotin, digoxigenin, fluorescein and dinitrophenol
• Enzyme labels such as alkaliine phosphatase, horseradish peroxidase and ß-galactosidase
• Fluorescent donor-acceptor pairs, fluorescer-quencher pairs
• Branched and linear multiply labeled arrays, linkers, particles, etc.
• Mixtures of two or more different labels

 

Multiple Labeling

Synthesis of one or both strands of a double stranded nucleic acid by LMO is capable of incorporating multiple labels via the use of labeled short oligomers. As long as the presence of the label on an oligomer does not interfere with ligation, many labels can be built into the growing strand(s). Spacing as close as one label every fifth base can be achieved ith pentamer ligation. Ligation efficiency is higher when labels are at nonterminal positions.

A representative process for preparing ds DNA in which each strand is uniquely labeled is illustrated below.

The use of LMO for labeling nucleic acid products finds application in diverse areas including preparing labeled probes, nucleic acid hybridization assays, sequence analysis, and gene expression studies. An example of the use of LMO for detecting a target nucleic acid by synthesis of a labeled complementary oligonucleotide is illustrated below. The process shown uses a combination of controlled labeling with a small hapten and post-synthesis labeling with a multi-label polymeric tag to increase sensitivity. Enzymatic turnover of the enzyme label provides additional signal amplification.
 

LMO Technology - Amplification

The simultaneous enzymatic ligation of a set of contiguous oligomer 5'-phosphates with a primer-template hybrid provides a new paradigm for linear and exponential amplification of nucleic acids. An amplification reaction contains a primer for each strand to be amplified and a set of downstream oligomers, each phosphorylated at it's 5' end, the template strand and a thermostable ligase.

• Each cycle of the thermocycling process consists of a strand separation step and an annealing/ligation step; the latter can be performed at one temperature or by using an intermediate annealing temperature as depicted below
• Cycles as short as 15 sec and up to 2 min in a standard thermal cycler
• Efficient amplification is accomplished in 30-40 cycles
• Amplification factors of at least 1 million result when performed in exponential mode

LMO amplification makes it possible to sequence-specifically amplify one allele of a genetic fragment with multiple alleles. Inclusion of an oligo spanning the polymorphic site which is specific for only one of the alleles permits only that allele to be replicated. Assays for disease related single nucleotide polymorphisms (SNPs) are easy to devise.
 

Amplification by LMO affords other capabilities which are not available in polymerase-based methods:

• The endpoints of the amplification product strand can be determined by the location of the primers or the location of the last consecutive oligomer or a combination of the two methods
• Many different configurations can be devised including the production of single or double overhangs or blunt end products
• One or both strands can be independently labeled; label spacing can be precisely controlled
• Multiple primers, as the 5'-phosphate, can be interspersed within a strand; bidirectional extension can be useful in amplifying regions with significant secondary structure
• Deliberate mutations can be designed into the amplified product using oligomers with internal mismatches

Amplification Configurations

The flexibility in the choice of direction of primer extension leads to numerous possible configurations for the amplification design. Since either strand can be prepared by 5' to 3' or 3' to 5' ligation, the two strands can be prepared by extension in the same sense or can differ. The first cycle of a novel design for LMO amplification conducted by 5' to 3' replication of one strand and 3' to 5' replication of the other is depicted below. In addition, the primers are shown offset to illustrate the ability to prepare amplicons with overhangs.



The ability to simultaneously ligate short oligomers to both termini of a hybridized primer oligonuleotide 5'-phosphate means that the primer need not be located at the end of the product strand. One or both strands can contain an internal primer sequence.



Still other designs can utiiize more than one primer within a strand. Primers can be located, e.g., at both ends of a strand to aid in the creation of defined overhangs. A primer might be located internally to aid in replicating past loci which form stem-loop structures. One of several potential examples is depicted below, only the first cycle being represented.

Rapid Amplification

LMO amplification of DNA templates with a thermostable ligase can be adapted to use on commercial thermocyclers. Amplification parameters are set in a manner familiar to users of PCR. A representative application of LMO amplification of a plasmid DNA template is described below.

A 68-base region of linearized pUC18 plasmid DNA and pUC285 plasmid DNA were amplified in a protocol using a pair of 20-base primers and six octamers per strand. Forty cycles of amplification used a temperature program of 94°C - 5 s; 55°C - 5 s; 35°C - 5 s. Amplified DNA was analyzed by chemiluminescent Southern blot. The image below is a 1 minute exposure made 10 minutes after adding the substrate.

Chemiluminescent Blot of LMO-Amplified DNA
15 second cycles

Lane 1 - Marker
Lane 2 - 1 ng pUC18
Lane 3 - 1 ng pUC285
Lane 4 - 100 pg pUC285
Lane 5 - Blank

The 68-base region of linearized pUC18 plasmid was also amplified in a protocol with longer cycle times using a pair of 20-base primers and six octamers per strand. Thirty cycles of amplification used a temperature program of 94°C - 30 s; 55°C - 30 s; 40°C - 30 s. Chemiluminescent Southern blot of the amplified product allowed detection of 0.01 pg of DNA template.

Chemiluminescent Blot of LMO-Amplified DNA
1.5 minute cycles

Lane M - Marker
Lane 1 - 0 fg pUC18
Lane 2 - 10 fg
Lane 3 - 100 fg
Lane 4 - 1 pg
Lane 5 - 10 pg
Lane 6 - 100 pg
Lane 7 - 1 ng

Ethidium-Stained Gel of LMO-Amplified DNA
1.5 minute cycles

Bidirectional Amplification

One of the unique features of LMO amplification is the ability to ligate a set of contiguous oligomer 5'-phosphates onto either end of a primer, provided the primer has a phosphate group on the 5'-end. We have found that under appropriate conditions, ligation can be made to occur at useful levels from either the 3' or 5' end of a primer or from both ends concurrently. Amplification by ligation in the 5' to 3' direction in the sense familiar to PCR users is shown here.



Amplification can be achieved in the 3' to 5' direction using a pair of primer 5'-phosphates as illustrated below. Ligation of sets of oligomers in a themocycling protocol produces an amplified polynucleotide where the primers are situated at the 3'-end of the product strands.



The flexibility in the choice of direction of primer extension leads to a myriad of configurations for the amplification design. Either strand can be prepared by 5' to 3' or 3' to 5' ligation or both directions at the same time. The two strands can be prepared by extension in the same sense or can differ.
 

Sequence Specific Amplification

LMO amplification makes it possible to sequence-specifically amplify and/or detect one allele of a gene or gene fragment that has multiple alleles. Use of an allele-specific oligomer spanning the polymorphic site permits only that allele to be replicated. In a model two allele system containing a deletion mutation and uniform size short oligomers, as shown below, the allelic genes are differentially replicated using either Set 1 or Set 2 oligomers.



Demonstrations of differential amplification and detection in assays for mutations are provided on the Assays section.

Differential detection can also be accomplished by amplifying both sequence variants together, each with a different label. Only one set of oligomers can ligate to an allele because of the requirement of contiguous alignment. When the polymorphism is a base substitution not involving differences in the length of the sequence, the diagnostic oligomers can be made different lengths so that the respective sets of "downstream" oligomers will only be in registration with one oligomer.



Although the mutation-specific oligomer is depicted as being immediately adjacent to the primer, in some applications it is useful to interpose additional short oligomers between the primer and the mutation-specific oligomer. Additional "universal" labeled oligomers can serve as a ligation or amplification internal control to signal the efficiency of the ligation process.