Conventional Sequencing Techniques Research Articles

Assessing a single-cell multi-omic analytic platform to characterize ex vivo-engineered T-cell therapy products.

Genetically engineered CD8+ T cells are being explored for the treatment of various cancers. Analytical characterization represents a major challenge in the development of genetically engineered cell therapies, especially assessing the potential off-target editing and product heterogeneity. As conventional sequencing techniques only provide information at the bulk level, they are unable to detect off-target CRISPR translocation or editing events occurring in minor cell subpopulations. In this study, we report the analytical development of a single-cell multi-omics DNA and protein assay to characterize genetically engineered cell products for safety and genotoxicity assessment. We were able to quantify on-target edits, off-target events, and potential translocations at the targeting loci with per-cell granularity, providing important characterization data of the final cell product. Conclusion: A single-cell multi-omics approach provides the resolution required to understand the composition of cellular products and identify critical quality attributes (CQAs).

ScLSTM: single-cell type detection by siamese recurrent network and hierarchical clustering

MotivationCategorizing cells into distinct types can shed light on biological tissue functions and interactions, and uncover specific mechanisms under pathological conditions. Since gene expression throughout a population of cells is averaged out by conventional sequencing techniques, it is challenging to distinguish between different cell types. The accumulation of single-cell RNA sequencing (scRNA-seq) data provides the foundation for a more precise classification of cell types. It is crucial building a high-accuracy clustering approach to categorize cell types since the imbalance of cell types and differences in the distribution of scRNA-seq data affect single-cell clustering and visualization outcomes.ResultTo achieve single-cell type detection, we propose a meta-learning-based single-cell clustering model called ScLSTM. Specifically, ScLSTM transforms the single-cell type detection problem into a hierarchical classification problem based on feature extraction by the siamese long-short term memory (LSTM) network. The similarity matrix derived from the improved sigmoid kernel is mapped to the siamese LSTM feature space to analyze the differences between cells. ScLSTM demonstrated superior classification performance on 8 scRNA-seq data sets of different platforms, species, and tissues. Further quantitative analysis and visualization of the human breast cancer data set validated the superiority and capability of ScLSTM in recognizing cell types.

Single-Cell Transcriptomics of Mtb/HIV Co-Infection.

Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) co-infection continues to pose a significant healthcare burden. HIV co-infection during TB predisposes the host to the reactivation of latent TB infection (LTBI), worsening disease conditions and mortality. There is a lack of biomarkers of LTBI reactivation and/or immune-related transcriptional signatures to distinguish active TB from LTBI and predict TB reactivation upon HIV co-infection. Characterizing individual cells using next-generation sequencing-based technologies has facilitated novel biological discoveries about infectious diseases, including TB and HIV pathogenesis. Compared to the more conventional sequencing techniques that provide a bulk assessment, single-cell RNA sequencing (scRNA-seq) can reveal complex and new cell types and identify more high-resolution cellular heterogeneity. This review will summarize the progress made in defining the immune atlas of TB and HIV infections using scRNA-seq, including host-pathogen interactions, heterogeneity in HIV pathogenesis, and the animal models employed to model disease. This review will also address the tools needed to bridge the gap between disease outcomes in single infection vs. co-infection. Finally, it will elaborate on the translational benefits of single-cell sequencing in TB/HIV diagnosis in humans.

The Succession of the Cellulolytic Microbial Community from the Soil during Oat Straw Decomposition.

The process of straw decomposition is dynamic and is accompanied by the succession of the microbial decomposing community, which is driven by poorly understood interactions between microorganisms. Soil is a complex ecological niche, and the soil microbiome can serve as a source of potentially active cellulolytic microorganisms. Here, we performed an experiment on the de novo colonization of oat straw by the soil microbial community by placing nylon bags with sterilized oat straw in the pots filled with chernozem soil and incubating them for 6 months. The aim was to investigate the changes in decomposer microbiota during this process using conventional sequencing techniques. The bacterial succession during straw decomposition occurred in three phases: the early phase (first month) was characterized by high microbial activity and low diversity, the middle phase (second to third month) was characterized by low activity and low diversity, and the late phase (fourth to sixth months) was characterized by low activity and high diversity. Analysis of amplicon sequencing data revealed three groups of co-changing phylotypes corresponding to these phases. The early active phase was abundant in the cellulolytic members from Pseudomonadota, Bacteroidota, Bacillota, and Actinobacteriota for bacteria and Ascomycota for fungi, and most of the primary phylotypes were gone by the end of the phase. The second intermediate phase was marked by the set of phylotypes from the same phyla persisting in the community. In the mature community of the late phase, apart from the core phylotypes, non-cellulolytic members from Bdellovibrionota, Myxococcota, Chloroflexota, and Thermoproteota appeared. Full metagenome sequencing of the microbial community from the end of the middle phase confirmed that major bacterial and fungal members of this consortium had genes of glycoside hydrolases (GH) connected to cellulose and chitin degradation. The real-time analysis of the selection of these genes showed that their representation varied between phases, and this occurred under the influence of the host, and not the GH family factor. Our findings demonstrate that soil microbial community may act as an efficient source of cellulolytic microorganisms and that colonization of the cellulolytic substrate occurs in several phases, each characterized by its own taxonomic and functional profile.

Genetic Spectrum and Characteristics of Hereditary Optic Neuropathy in Taiwan.

Hereditary optic neuropathy (HON) is a group of genetically heterogeneous diseases that cause optic nerve atrophy and lead to substantial visual impairment. HON may present with optic nerve atrophy only or in association with various systemic abnormalities. Although a genetic survey is indispensable for diagnosing HON, conventional sequencing techniques could render its diagnosis challenging. In this study, we attempted to explore the genetic background of patients with HON in Taiwan through capture-based next-generation sequencing targeting 52 HON-related genes. In total, 57 patients from 48 families were recruited, with 6 patients diagnosed as having Leber hereditary optic neuropathy through initial screening for three common variants (m.3460G>A, m.11778G>A, m.14484T>C). Disease-causing genotypes were identified in 14 (33.3%) probands, and OPA1 variants were the most prevalent cause of autosomal HON. Exposure to medications such as ethambutol could trigger an attack of autosomal dominant optic atrophy. WFS1 variants were identified in three probands with variable clinical features in our cohort. Hearing impairment could occur in patients with OPA1 or WFS1 variants. This is the first comprehensive study investigating the genetic characteristics of HON in Taiwan, especially for autosomal HON. Our results could provide useful information for clinical diagnosis and genetic counseling in this field.

Accurate prediction of RNA 5-hydroxymethylcytosine modification by utilizing novel position-specific gapped k-mer descriptors

RNA modification is an essential step towards generation of new RNA structures. Such modification is potentially able to modify RNA function or its stability. Among different modifications, 5-Hydroxymethylcytosine (5hmC) modification of RNA exhibit significant potential for a series of biological processes. Understanding the distribution of 5hmC in RNA is essential to determine its biological functionality. Although conventional sequencing techniques allow broad identification of 5hmC, they are both time-consuming and resource-intensive. In this study, we propose a new computational tool called iRNA5hmC-PS to tackle this problem. To build iRNA5hmC-PS we extract a set of novel sequence-based features called Position-Specific Gapped k-mer (PSG k-mer) to obtain maximum sequential information. Our feature analysis shows that our proposed PSG k-mer features contain vital information for the identification of 5hmC sites. We also use a group-wise feature importance calculation strategy to select a small subset of features containing maximum discriminative information. Our experimental results demonstrate that iRNA5hmC-PS is able to enhance the prediction performance, dramatically. iRNA5hmC-PS achieves 78.3% prediction performance, which is 12.8% better than those reported in the previous studies. iRNA5hmC-PS is publicly available as an online tool at http://103.109.52.8:81/iRNA5hmC-PS. Its benchmark dataset, source codes, and documentation are available at https://github.com/zahid6454/iRNA5hmC-PS.

Metagenomics Approaches in Discovery and Development of New Bioactive Compounds from Marine Actinomycetes.

Marine actinomycetes are prolific sources of marine drug discovery system contributing for several bioactive compounds of biomedical prominence. Metagenomics, a culture-independent technique through its sequence- and function-based screening has led to the discovery and synthesis of numerous biologically significant compounds like polyketide synthase, Non-ribosomal peptide synthetase, antibiotics, and biocatalyst. While metagenomics offers different advantages over conventional sequencing techniques, they also have certain limitations including bias classification, non-availability of quality DNA samples, heterologous expression, and host selection. The assimilation of advanced amplification and screening methods such as φ29 DNA polymerase, Next-Generation Sequencing, Cosmids, and recent bioinformatics tools like automated genome mining, anti-SMASH have shown promising results to overcome these constrains. Consequently, functional genomics and bioinformatics along with synthetic biology will be crucial for the success of the metagenomic approach and indeed for exploring new possibilities among the microbial consortia for the future drug discovery process.

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.

BackgroundTargeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.MethodWe used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.ResultsBoth FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.ConclusionTargeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

New insights into genotype and phenotype of VWD

AbstractRecent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype–phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.

New insights into genotype and phenotype of VWD.

Recent advances in VWD research have improved our understanding of the genotype and phenotype of VWD. The VWF gene is highly polymorphic, with a large number of sequence variations reported in healthy individuals. This can lead to some difficulty when attempting to discern genotype-phenotype correlations because sequence variations may not represent disease. In type 1 VWD, mutations can be found throughout the VWF gene, but likely pathogenic sequence variations are found in only ∼2/3 of type 1 VWD patients. Sequence variations in type 2 VWD are located in the region corresponding to the defect in the VWF protein found in each type 2 variant. In type 3 VWD, sequence variations are not confined to a specific region of the VWF gene and also include large deletions that may not be picked up using conventional sequencing techniques. Use of genetic testing may be most helpful in diagnosis of type 2 VWD, in which a larger number of known, well characterized mutations are present and demonstration of one of these may help to confirm the diagnosis. Bleeding symptoms in general are more severe with decreasing VWF levels and more severe in type 2 and type 3 VWD compared with type 1 VWD. Prediction of phenotype for an individual patient, however, is still difficult, and the addition of genetic data will be most helpful in ascertaining the correct diagnosis for VWD patients.

DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm 9 mouse reference genome assembly contains at least 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments have persisted through several iterations of the mouse reference genome and have been difficult to detect using conventional sequencing techniques. The ability to map SCE events at high resolution and fine-tune reference genomes by Strand-seq dramatically expands the scope of single-cell sequencing.

Abstract 3: HER2 gene-amplified human breast cancer cells harboring a gatekeeper T768M mutation in HER2 overexpress EGFR ligands and are sensitive to dual therapeutic blockade of EGFR and HER2

Abstract Intrinsic or acquired mutations in receptor tyrosine kinases (RTKs) can confer resistance to RTK-targeted therapies. A previous study reported that a ‘gatekeeper’ T768M mutation in HER2 confers resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We generated BT474 cells stably expressing either T768M HER2 or GFP. Treatment with lapatinib disrupted HER3-p85 complex and blocked phosphorylation of HER2, HER3, AKT and Erk1/2 in BT474-GFP but not in BT474-T768M cells. BT474-T768M cells were also resistant to other anti-HER2 therapies such as trastuzumab. BT474-T768M cells were sensitive to the PI3K inhibitor BKM120 but not the MEK inhibitor CI1040, suggesting they still depend on the PI3K pathway for survival. Treatment with the irreversible HER2/EGFR inhibitor BIBW2992 (afatinib) eliminated P-HER2 and inhibited growth of cells expressing the mutant, suggesting they still depend on the ErbB receptor network to activate PI3K. Indeed, like in BT474-GFP (control) cells, BIBW2992 disrupted the association of p85 (PI3K) with HER3 and blocked phosphorylation of AKT. A co-culture experiment with varying proportions of BT474-GFP and BT474-T768M cells revealed that lapatinib-resistant acini emerge when as low as 5% of the population has T768M HER2. Moreover, we found that only 3.2% of the HER2 alleles had the T768M mutation in the BT474-T768M cells. This result suggests that allelic dilution will render it difficult to identify this drug resistant mutation with conventional sequencing techniques (i.e., Sanger) in patients with HER2 gene-amplified breast cancer. Next, we stably expressed either wild-type or T768M HER2 in MCF10A cells. Both WT and T768M HER2 resulted in increased phosphorylation of HER3 and AKT. MCF10A-HER2 but not MCF10A-T768M cells were sensitive to growth inhibition by lapatinib. Lapatinib blocked HER2, HER3 and tyrosine phoshorylation in MCF10A-HER2 but not in MCF10A-T768M cells. Quantitative PCR indicated that the BT474-T768M cells produce more EGFR ligands (EGF, TGFα, amphiregulin and HB-EGF) compared to BT474-GFP cells. Treatment with cetuximab, an EGFR antibody that blocks ligand binding, restored sensitivity of BT474-T768M cells to trastuzumab. Further, the BT474-T768M cells were sensitive to a combination of trastuzumab and lapatinib both in vitro and in vivo. Hence, dual blockade of HER2 and EGFR might be effective at trumping the development of HER2-overexpressing cancers bearing a gatekeeper mutation in T768M. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3. doi:1538-7445.AM2012-3

Acquired Imatinib Resistance in CML Is Associated with TKD Mutations of the BCR-ABL Fusion Gene and/or Additional Chromosomal Aberrations: Certain TKD Mutations but Not TP53 Mutations Are Associated with Chromosome Instability.

Acquired resistance to imatinib is an important issue in CML. It was correlated to mutations in the tyrosine kinase domain (TKD) of the BCR-ABL fusion gene in 30–80% of cases. However, the mechanisms of resistance in cases without TKD mutations are still insufficiently understood. One potential further mechanism are additional chromosomal abnormalities (ACA) secondary to the Philadelphia translocation. We hypothezised that patients without TKD mutations might have a higher incidence of ACAs. Thus the aim of our analysis was to evaluate correlations between these two genetic mechanisms. In total 88 CML patients with acquired imatinib resistance were analyzed in parallel for additional chromosomal abnormalities by karyotyping and for resistance mutations in the TKD of the BCR-ABL fusion gene by sequence analysis. In 40 of the 88 pts (45.5%) at least one resistance mutation was detected. Of the 40 mutated cases 6 (15%) revealed two different resistance mutations whereas in 48 pts (54.5%) no mutation was detected with a sensitivity of 10–20% that is typical for conventional sequencing techniques. In the group not affected by TKD mutations 15 of 48 (31.3%) cases showed ACAs (−Y: n=2, t(3q26): n=4; +8: n=4; +Ph: n=5; del(17p): n=2; nonrecurrent reciprocal translocations: n=4; complex aberrant: n=1; others: n=2). 8 of these 15 cases had more than one ACA (median 3; range 2–7). In the group with resistance mutations a nearly equal number of 15/40 (37.5%) cases revealed ACAs and the spectrum of aberrations was very similar: (−Y: n=1, t(3q26): n=3; +8: n=5; +Ph: n=3; del(17p): n=2; complex aberrant: n=2; others: n=5). In this group 7 cases had more than one ACA (median:3; range: 2–3). Thus the total amount of pts with ACAs, the spectrum of ACA as well as the number of ACAs per patient is nearly equal in the mutated and the unmutated cohort. In the 40 TKD mutated cases 16 different mutations were detected (M244V: n=2 ; L248V: N=1; G250E: n=6; Y253H: n=3; E255K: n=2; D276G: n=1; E279K: n=2; L298V: n=1; L298V: n=1; F311I: n=1; F311L: n=1; T315I: n=11; F317L: n=1; F359C: n=3; F359I: n=1; M351T: n=6; H396R: n=4;). Most of the recurrent mutations are distributed equally in the groups with or without ACAs. Solely in cases with M244V, G250E or Y253H we never observed ACAs. Thus among 12 cases within the region of amino acid 244–253 there is no case with any ACA indicating that mutations within this region are strong enough to cause high resistance without further need of any ACA or alternatively are caused by different mechanisms that are not based on further genetic instability. In comparison 5 of 11 cases with T315I and 3 of 4 with H396R mutations are associated with multiple or complex chromosomal aberrations (p=0.014). Five pts were analyzed with both methods at 2–4 time points under dose escalation of imatinib. Two cases had stable G250E mutations without ACA for 2 and 10 months. One pt first revealed a T315I mutation and developed two reciprocal tranlocations t(5;10) (q31;q24) and t(6;17)(q26;q12) in addition to the T315I within 6 months. A second pt first had an E279K mutation and within one year developed an additional Y253H mutation and a +8. A third pt first had a t(3;21), after 2 months developed an additional T315I, and after further 5 months an additional F359C mutation. These few cases show that ACAs can preceed TDK mutations and vice versa. Although evolvement of resistance mainly is believed to be a process of selection some degree of genetic instability should be underlying to create some genetic diversity as a pool for any selection mechanism. Mutations within the TP53 gene have been associated with genetic instability and complex aberrant karyotypes in AML as well as in CLL. To analyze whether TP53 may also be involved in the genetic instability that leads to imatinib resistance we sequenced exons 4–9 of TP53 of 16 TKD mutated cases from this cohort (8 with complex aberrations and 8 without ACAs). No TP53 mutation was detected in all 16 cases indicating that TP53 is unlikely a major player in the development of resistance to imatinib. In conclusion,ACA are equally distributed between TKD mutated and unmutated cases.amino acid exchanges in the region 244–253 are not observed together with ACA whereas T315I and F359C are associated with multiple aberrations.TP53 mutations probably do not play a role in the development of genetic changes that are associated with imatinib resistance.

Multiplexed Assays for Detection of Mutations in PIK3CA

Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.

Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey.

BackgroundExtensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA.ResultsWe sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis).ConclusionThis approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.

Amino Acid Sequence Analysis, Gene Construction, Cloning, and Expression of Gelonin, a Toxin Derived fromGelonium multiflorum

The plant toxin gelonin is an extremely potent inhibitor of protein synthesis, similar in action to ricin. The mature protein primary sequence was obtained using conventional sequencing techniques. Gelonin was found to be composed of 258 amino acids and contains 21 lysine residues. This toxin shares approximately 33% sequence homology with trichosanthin and ricin A chain. A 774 bp synthetic gene encoding gelonin was synthesized and expressed in E. coli. Recombinant gelonin (approximately 28 kD) expression was monitored and demonstrated by western analysis. Purification and functional activity studies demonstrated that this protein behaves identically to that of the natural product. Recombinant gelonin (RG) thus joins a growing list of recombinant toxins currently available for use in the construction of recombinant immunotoxins composed of gelonin fused to binding domains of antibodies, growth factors, or other cytokines.

MUTATIONS IN THE P53 GENE AT CODON 249 ARE RARE IN SQUAMOUS-CELL CARCINOMA OF THE HEAD AND NECK

Mutations in the p53 tumour suppressor gene are common in a wide range of human malignancies. In particular G-T transversions in the p53 gene occur at a high frequency in lung cancer tumours and as the same mutagens maybe responsible for p53 mutations in head and neck squamous cell carcinomas, a preliminary investigation was carried out to establish if this may be the case. Codon 249 of the p53 gene has been found to be a hot spot of G-T transversions in hepatocellular carcinomas, thus it was decided to initially investigate this site using specific primers and restricting the PCR product with the HaeIII restriction endonuclease. Fifty seven squamous cell carcinomas of the head and neck were investigated using this technique and no mutations were found at codon 249 in the p53 gene. In this study twenty six of the specimens were also examined immunohistochemically using a number of antibodies to p53 (PAb 421, PAb 1801 and CM-1), and 19 were found to have positive staining. Moreover as positive p53 staining is considered to be synonymous with p53 mutations, over two thirds of our earlier immunohistochemical study indicated p53 mutations in head and neck tumours. The technique used in this paper has the advantage of scanning a large number of tumours for mutations at suspect sites and maybe of use in the future if specific mutational hot spots are found in the p53 gene in head and neck cancers. In the meantime p53 mutations in these tumours are been investigated by conventional sequencing techniques.