# Heteroduplex thermostable ligation assembly: a new platform to rapidly generate large DNA molecules

> **NIH NIH R41** · CHESAPEAKE GENOMIC SYSTEMS LLC · 2021 · $256,645

## Abstract

In vitro assembly of DNA fragments >20 kb has recently identified as a critical technical challenge for writing
synthetic genomes. Meeting this challenge will increase the efficiency of genome writing projects that depend
on large synthetic DNAs, which are iteratively recombined to replace chromosome regions in a step-wise manner
to partially or completely re-write bacterial or yeast chromosomes. The long-term goal of this project is to develop
an efficient, low-cost strategy to generate completely synthetic large DNAs, from >50 kb up to 1 Mbp, without
homologous recombination in yeast or bacteria. The objective of the work proposed here is to optimize the
parameters of this novel approach we term Heteroduplex Thermostable Ligase Assembly (HTLA) to generate
large DNAs completely in vitro. The rationale for this work is that successful completion will provide proof-of-
concept that heteroduplex cloning can be used to rapidly generate large DNAs in a low-cost, commercializable
manner. To accomplish these goals within the budget, PCR-generated bacteriophage lambda genome fragments
will be used as a paradigm for de novo synthesized DNAs. Two HTLA strategies to generate large DNAs will be
systematically explored by achieving the goals of three related but independent Specific Aims: 1) To determine
optimal HTLA conditions to generate ~2 kb fragments with ligation-ready sticky ends for assembly of a large
DNA construct. One-cycle HTLA reaction conditions to join 1 kb lambda genome parts to generate ~2 kb sticky
end blocks (SEBs) will be systematically varied to maximize yield. DNA part overlap length will also be optimized.
Sticky end length on SEBs will then be varied to achieve optimal ligation with T4 or 9°N™ DNA ligase. SEBs will
be ligated in increasing numbers to determine the practical limit of final product length. Sequence fidelity of
assembled sequences will be determined; 2) To determine the maximum number of ~1 kb heteroduplex DNA
molecules that can be reproducibly joined in a one-pot, one-step method with high sequence fidelity by HTLA
and Cyclic Heteroduplex Thermostable Ligation Assembly (CHTLA). One kb lambda genome parts in increasing
numbers will be joined using HTLA to determine the upper size limit for SEB generation with a yield sufficient for
subsequent steps. The effect of DNA part overlap length (100-500) on reaction efficiency will be determined. 10-
cycle CHTLA reactions will also be performed with increasing numbers of 1 kb lambda genome parts to probe
the upper limit on final product length that can be generated by CHTLA. Sequence fidelity will be determined; 3)
To use optimized heteroduplex cloning strategies identified in Aims 1 and/or 2 to seamlessly generate a complete
lambda phage genome that can be packaged into infectious particles. Using optimized conditions determined in
Aim 1 and 2, a completely assembled lambda genome will be generated, packaged, and used to infect E. coli.
Successful completion of this Phase 1 pro...

## Key facts

- **NIH application ID:** 10383266
- **Project number:** 1R41GM145048-01
- **Recipient organization:** CHESAPEAKE GENOMIC SYSTEMS LLC
- **Principal Investigator:** CHARLES J. BIEBERICH
- **Activity code:** R41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $256,645
- **Award type:** 1
- **Project period:** 2021-09-23 → 2023-09-22

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10383266

## Citation

> US National Institutes of Health, RePORTER application 10383266, Heteroduplex thermostable ligation assembly: a new platform to rapidly generate large DNA molecules (1R41GM145048-01). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10383266. Licensed CC0.

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