How to Read and Interpret Dna Chromatography
A typical electropherogram from a single PCR product is shown below. Each peak represents a unmarried nucleotide in the DNA sequence, and each nucleotide has a different color; A is green, T is red, C is blue and G is black.
From Lee, S.H.; Vigliotti, J.S.; Vigliotti, V.S.; Jones, Westward.; Moorcroft, T.A.; Lantsman, K. DNA Sequencing Diagnosis of Off-Season Spirochetemia with Low Bacterial Density in Borrelia burgdorferi and Borrelia miyamotoi Infections. Int. J. Mol. Sci. 2014, 15, 11364-11386.
The average PCR product contains 200 nucleotides of sequencing, and the maximum length that tin can be sequenced by the Sanger method is about 600 nucleotides. The amount of data generated past Sanger sequencing for each patient therefore varies depending on the size of the gene beingness sequenced. Sequencing of a relatively small gene such as HNF1A generates over 2500 nucleotides of sequencing data per patient, and since a mutation can occur at any of the nucleotides within the electropherogram, each nucleotide requires checking in turn for the presence of a mutation.
Sanger sequencing analysis is performed on a comparative basis, where the patient's electropherogram is compared against an electropherogram from a Deoxyribonucleic acid sample without a mutation. Any observed differences between the two traces are recorded and analysed for their potential pathogenic upshot on the protein.
Historically this was performed past a visual comparing of each nucleotide pinnacle in the two traces, but this is time consuming, prone to error and cannot run across the workload and quality demands of today's modernistic, loftier throughput diagnostic laboratory.
Today, sequencing analysis is performed past software that tin can perform comparative analysis of tens of thousands of nucleotides within seconds. The software automatically detects mutations and provides a description of the mutation at the Deoxyribonucleic acid and poly peptide level with a high degree of accuracy and sensitivity.
Visual inspection: time consuming and error prone 
Software analysis: fast and accurate 
Created by Mutation Surveyor five iv.0.6 SoftGenetics, Land College PA United states of america 16803
What was the result of the Sanger sequencing analysis of Dan'south HNF1A gene? The output from the sequencing analysis software can be seen in the picture show above. Information technology shows Dan's DNA sequence with a heterozygous missense mutation in exon vii of the HNF1A gene. The mutation is the substitution of a C nucleotide with a T nucleotide on one copy of his HNF1A gene (indicated past the presence of a C and T peak at nucleotide position 1340 in Dan's DNA sequence). This results in a change in the amino acid (from proline to leucine) at amino acrid number 447 in the HNF1A protein sequence.
HNF1A is a transcription factor. Its function is to control the expression of other genes in the beta cell of the pancreas that are involved in insulin product and secretion, including the insulin gene itself. Transcription factors work by bounden to the promoters of genes and activating gene transcription (the process of making mRNA copies of the factor to be used for protein synthesis). The promoter is the switch that turns the gene on or off, and the transcription factor is the finger that operates that switch.
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Dan's mutation occurs within the transactivation domain of the HNF1A protein; the region of the protein responsible for flicking the switch. The mutant HNF1A poly peptide is able to bind to the promoter, merely cannot turn on gene expression. This results in defective expression of genes required for insulin production and secretion, which results in insulin deficiency and diabetes. This is a known pathogenic mutation and we can therefore exist confident that the event confirms a diagnosis of HNF1A MODY in Dan.
Source: https://www.futurelearn.com/info/courses/diabetes-genomic-medicine/0/steps/10061
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