Homopolymer Tail Mediated Ligation PCR

HOMOPOLYMER TAIL MEDIATED LIGATION PCR

Inventors: David Lazinski and Andrew Camilli

Tufts Case # T001804

Invention

Drs. David Lazinski and Andrew Camilli, Tufts University School of Medicine, have developed a new molecular method for amplifying DNA molecules of both known and unknown sequence, termed "homopolymer tail mediated ligation PCR" (HTML-PCR) with important research, diagnostic and forensic applications where the supply or integrity of sample DNA may be limited. HTML-PCR can be used to attach to amplified DNA-defined (universal) end sequences to be used for downstream applications such as massively parallel sequencing (MPS) (a.k.a. next-generation sequencing), traditional sequencing, purification, non-covalent or covalent attachment to other molecules including DNA (cloning), immobilization, hybridization, binding assays, transformation, etc. The HTML-PCR method can be used to process miniscule amounts of starting DNA material, amounts that are below that needed for adapter ligation protocols, and is effective even when only 100 picograms (as low as 10 picograms worked) of sample DNA is used (approximately equal to the amount of DNA in two human cells). HTML-PCR functions with starting DNA amounts that can vary by up to five orders of magnitude and with template molecules of no minimum length. HTML-PCR has been demonstrated in proof-of-principle experiments to construct MPS libraries starting from nanogram and picogram quantities of Vibrio cholerae and Streptococcus pneumoniae genomic DNA.

Problems addressed by the invention

1. In many experimental, diagnostic, and clinical settings, it can be difficult or impossible to obtain sample DNA in quantities sufficient to allow for its cloning and/or sequencing using existing methods. HTML-PCR is able to amplify any DNA of unknown sequence even when it is present in minute quantities or when its integrity has been compromised. The extent of amplification is sufficient to enable any desired downstream application.
2. Even when sample DNA is abundant, a series of labor-intensive steps are required to convert it to a form suitable for MPS. Compared to current methods for library sample preparation for MPS, HTML-PCR is much more efficient, does not produce inhibitory adapter dimers, does not require gel purification, is less labor intensive and is far better suited to high-throughput and robotic methodologies. Sample preparation cost will be much lower with HTML-PCR compared to existing methods.

Existing solutions to the problems

Whole genome amplification. When sample DNA is in limited supply, the current method of choice for its amplification is known as Multiple Displacement Amplification. In an isothermal amplification reaction, this method uses a highly processive DNA polymerase isolated from bacteriophage phi29 that has exceptional strand displacement properties. The method works best with circular templates and/or with very long linear templates. Under these optimal conditions the level of amplification can reach 1,000-fold. However, as template size is reduced the method becomes increasingly inefficient. The Multiple Displacement Amplification method is not useful with partially degraded DNA templates where the average fragment length is less than a few hundred base-pairs.

MPS library construction. Current methods of construction of sample libraries for MPS involve the ligation of duplex or partial duplex DNA adapters to sample DNA in a method that we refer to as Adapter-Mediated Construction. For example, Illumina uses an Adapter-Mediated Construction strategy with a partial duplex adapter in which their sequence X is present on one strand of the adapter while their Y sequence is present on the opposite strand. A constant sequence C is present at the 3’ end of one strand and its complement, C’, is present on the 5’ end of the opposite strand. Hybridization of C to C’ creates the partial duplex adapter and simultaneously creates a single dT 3’ overhang. Sample DNA is fragmented and treated with a series of enzymatic steps such that it contains a single dA 3’ overhang. Annealing of the adapter dT overhang to the sample DNA dA overhang enables a ligation event mediated by T4 DNA ligase. The product of ligation, 5’ XC-sample DNA-C’Y 3’ is then amplified in a PCR reaction that uses X and Y sequence specific primers. While effective, this method has a number of disadvantages including:

(a) It requires a relatively large amount of starting DNA material;

(b) The interaction of the dT overhang with the dA overhang is very weak and consequently, the resulting ligation is very inefficient;

(c) An unintended and unwanted side reaction can occur when two adapter molecules are ligated to create an adapter dimer. As the adapter dimer is an ideal template for PCR, when present in sufficient quantities, it can out-compete with the amplification of the sample library; and

(d) To reduce the problem of adapter dimers it is necessary to gel purify adapter-ligated sample DNA from adapter dimers prior to the PCR reaction. Gel purification is very labor-intensive and serves as an impediment to high-throughput and robotic methods of library construction.

Advantages of HTML-PCR

Table 1 below compares HTML-PCR with the Illumina Adapter-Mediated Construction protocol for preparing sample libraries for MPS. HTML-PCR provides the following significant improvements over the Ilumina Adapter-Mediated Construction method, and other methods.

For whole genome amplification, even with ideal templates for Multiple Displacement Amplification, HTML-PCR can easily exceed the 1,000-fold amplification achieved by that method. Furthermore, for non-ideal templates degraded to 100-300 base pair lengths, while Multiple Displacement Amplification will not work, HTML-PCR will be fully functional. The ability to amplify partially degraded DNA is a major advantage of HTML-PCR and can enable the retrieval of sequences from samples where that was previously not possible.

For library construction for MPS, HTML-PCR suffers from none of the disadvantages associated with Adapter-Mediated Construction. While Adapter-Mediated Construction requires relatively large amounts of starting DNA sample material, HTML-PCR works with minute quantities. While Adapter-Mediated Construction ligation is very inefficient and is mediated by an extremely weak single dA:dT pairing, HTML-PCR ligation is very efficient and is mediated by the very stable interaction of seven consecutive dC:dG pairs. Furthermore, since HTML-PCR does not use adapters, no inhibitory adapter dimers are generated and there is no need for gel purification at any stage in the procedure. Compared with Adapter-Mediated Construction, HTML-PCR is less time consuming, less labor-intensive, more cost-effective and far better suited to high-throughput and robotic methodologies.

Additional Applications

• In addition to preparing genomic DNA samples for MPS (discussed above), another example application would be PCR amplification and sequencing of the junctions (insertion sites) of human LINEs and SINEs, which have great diagnostic (e.g. forensic, paternity, etc.) value because there exists numerous differences in insertion sites of LINEs and SINEs between individuals, even between siblings (though not between monozygotic twins).
• Medical diagnostic labs could use it in combination with DNA sequencing to identify specific pathogens in patients.
• Human genetics labs could use HTML-PCR to identify genetic lesions in patients or fetal tissue.
• Food safety laboratories could use the method to detect and identify food-borne pathogens.

Investigators

David Lazinski, Ph.D.; Andrew Camilli, Ph.D., Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University School of Medicine

Key Publication

Lazinski and Camilli, Biotechniques (2013), Jan; 54(1):25-34

Seed KD, Lazinski DW, Calderwood SB, Camilli A. Nature (2013) 494: 489-491

Klein BA, Tenorio EL, Lazinski DW, Camilli A, Duncan MJ, Hu LTBMC Genomics (2012) Oct 31; 13:578

Intellectual Property

US Patent 9,914,950 (March 13, 2018)

Illumina

sample prep

HTML-PCR

sample prep

Nextera sample prep

Sensitivity (minimum starting amount of DNA)

1,000- 5,000 nanograms DNA

100picograms-1 microgram DNA

1,000-50,000 picograms

Works with damaged DNA

No

Yes

No

Ligation efficiency

Poor

Excellent

N/A

Adapter dimers formed

Yes

No

No

Gel purification required

Yes

No

No

Compatible with high throughput/robotics

No

Yes

Yes

Accurate estimation of DNA concentration needed

Yes

No

Yes