Genomics
Sanger Sequencing
Overview – Sanger sequencing?
Sanger sequencing, or chain-termination sequencing, has
been a key method in DNA sequencing since the 1970s. It works by using labeled chain-terminating nucleotides incorporated by DNA polymerase during replication. This technique has greatly advanced functional and comparative genomics, evolutionary genetics, and complex disease studies. It was crucial in sequencing the first human genome in 2002. Due to its accuracy in validating cloning experiments and PCR fragments, Sanger sequencing continues to be a popular and essential tool in laboratories globally.
Applications – What are the advantages of Sanger sequencing?
Sanger DNA sequencing is widely used for research purposes like:
- Analyzing smaller genomic regions across numerous samples
- Sequencing variable genomic regions
- Confirming findings from next-generation sequencing (NGS) studies
- Verifying plasmid sequences, inserts, and mutations
- Conducting HLA typing
- Genotyping microsatellite markers
- Identifying single genetic variants responsible for diseases
Workflow – Sanger sequencing methods & technologies:
Dideoxy sequencing is a method for synthesizing complementary DNA strands based on a template DNA sequence. This technique employs both standard deoxynucleoside triphosphates (dNTPs) and modified dideoxynucleoside triphosphates (ddNTPs). The ddNTPs are chemically altered with fluorescent labels and a modification that halts DNA synthesis by preventing phosphodiester bond formation. As a result, DNA polymerase stops extending the strand each time a ddNTP is incorporated. The resulting DNA fragments are separated by capillary electrophoresis, where they traverse a gel-like matrix at different speeds according to their size. Each ddNTP is tagged with a distinct fluorescent dye, and the emitted fluorescence identifies the nucleotide sequence of the original DNA template.
Sanger sequencing vs. next-generation sequencing:
The field of DNA sequencing continued to advance beyond the development of Sanger sequencing, with next-generation sequencing (NGS) and third-generation technologies providing significant improvements over the traditional dideoxy method. Despite these advances, the chain-termination method is still widely utilized due to its distinct benefits. In particular, Sanger sequencing is often favored over NGS for its accuracy, longer read lengths, and lower error rates in sequencing individual DNA fragments, making it a preferred choice for certain applications.
- Sequencing individual genes
- Cost-effective analysis of single samples
- Verification of site-directed mutagenesis or cloned inserts
- Analysis of longer DNA fragments, up to approximately 1,000 base pairs
- Potentially fewer errors compared to next-generation sequencing (NGS) in certain scenarios
Nevertheless, next-generation sequencing is often considered to be superior to Sanger
sequencing, especially for project objectives that require:
- Cost-effective, simultaneous analysis of over 100 genes
- Discovery of novel variants by expanding the number of targets per sequencing run
- Analysis of samples with limited DNA input
- Sequencing entire genomes, particularly microbial genomes
Clinical applications of Sanger sequencing:
Sanger sequencing remains the most accurate form of DNA sequencing. It is still widely
used in clinical laboratories for the following applications:
- Diagnostic sequencing of a single gene.
- Testing for specific familial sequence variants, including:
1. Predictive genomic testing for at-risk relatives (e.g., assessing a familial BRCA1 variant associated with breast cancer risk).
2. Carrier testing for parents of a child with an autosomal recessive condition (e.g., cystic fibrosis); prenatal testing for known familial variants; and segregation analysis to help interpret the pathogenicity of a variant (e.g., determining if a variant is present in both an affected sibling and the proband). - Confirming variants identified through next-generation sequencing (NGS).
- Addressing gaps in NGS data.
Advantages and limitations of Sanger sequencing
Advantages
- Gold standard method for accurate detection of single nucleotide variants and small
insertions/deletions. - More flexible for testing for a specific familial variant than NGS.
- Cost effective where single samples need to be tested very urgently, so they cannot be batched up (for example, in prenatal testing, or parental carrier testing during a
pregnancy). - Less reliant on computational tools than NGS.
- In some cases, longer fragments (up to approximately 800-900 bp) can be sequenced than in short read NGS.
Limitations
- Limited throughput.
- Not cost effective for sequencing many genes in parallel, or for sequencing the same
region in many samples. - May not detect mosaicism.
- Can require a larger amount of input DNA than NGS.
Key messages
Sanger sequencing is a fast
and cost effective way of
reading the sequence of small targeted regions of the genome.
Sanger sequencing is the gold standard method for accurate detection of single nucleotide variants and small insertions/deletions.
Sanger sequencing is widely used to test for known familial variants, for validation of results obtained through NGS and for some single gene sequencing.or more routine small-scale projects and NGS is applied to meet large-scale sequencing needs.
Sanger sequencing is the gold standard method for accurate detection of single nucleotide variants and small insertions/deletions.
Sanger sequencing is widely used to test for known familial variants, for validation of results obtained through NGS and for some single gene sequencing.or more routine small-scale projects and NGS is applied to meet large-scale sequencing needs.
