Kyle W. Langford

Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pores constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42–53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing.

Decoding long nanopore sequencing reads of natural DNA

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA.

Significance Cells attach a methyl group (—CH3) to certain cytosines in DNA to control gene expression. These methylation patterns change over time and can be related to cell differentiation and diseases such as cancer. Existing methylation detection techniques are not ideal for clinical use. We pulled single-stranded DNA molecules through the biological pore MspA and found that ion currents passing through the pore reveal the methylation sites with high confidence. Hydroxymethylation, which differs from methylation by only one oxygen atom, also produces distinct signals. This technique can be developed into a research tool and may ultimately lead to clinical tests. Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.

Unsupported planar lipid membranes formed from mycolic acids of Mycobacterium tuberculosis

The cell wall of mycobacteria includes a thick, robust, and highly impermeable outer membrane made from long-chain mycolic acids. These outer membranes form a primary layer of protection for mycobacteria and directly contribute to the virulence of diseases such as tuberculosis and leprosy. We have formed in vitro planar membranes using pure mycolic acids on circular apertures 20 to 90 μm in diameter. We find these membranes to be long lived and highly resistant to irreversible electroporation, demonstrating their general strength. Insertion of the outer membrane channel MspA into the membranes was observed indicating that the artificial mycolic acid membranes are suitable for controlled studies of the mycobacterial outer membrane and can be used in nanopore DNA translocation experiments.

Continuously Scanning DNA with Nanopore MspA

Nanopore sequencing is a promising next-generation technology that is being enabled by the biological nanopore MspA. In this sequencing technique, current is driven through the ∼1 nm constriction of MspA. Single stranded DNA molecules are first drawn into the pore by an applied voltage. Next, a polymerase or helicase moves the DNA by discrete steps as it interacts with the pore. As the DNA moves through the pore, different nucleobases modulate the pores conductance to varying extents, allowing for extraction of sequence information from the current trace. In this experiment, we gain more complete information by using a variable applied voltage to stretch the DNA within the pore. Changing the voltage continuously repositions the DNA in the constriction. Coupling this voltage-induced movement with the enzyme-induced motion we reconstruct a profile characteristic of the DNA as it is pulled continuously through the constriction. This profile provides a tool for improving the de novo sequencing accuracy of the nanopore technique.