Genetic Analysis Discussion Example | PCR, Electrophoresis, DNA Sequences, Mutation Assessment, and Sources of Error

DNA was extracted from three plant-derived samples, and the target gene region was amplified by PCR. DNA concentrations of Samples A, B, and C were 42.5, 36.8, and 51.2 ng/µL, respectively.

Sample A had an A260/A280 ratio of 1.82 and an A260/A230 ratio of 2.03, and its purity was judged suitable for PCR.

Electrophoresis showed main bands of approximately 520 bp in Samples A and B. Sample C showed a band of approximately 700 bp.

The positive control showed the expected band of approximately 520 bp, while no main band was observed in the negative control.

Sequence analysis of the Sample A amplicon allowed comparison of 472 bases, of which 468 matched the reference sequence. Sequence identity was 99.15%.

Three base substitutions and one single-base deletion were observed.

Based on the band patterns, Sample A was estimated to be a short-allele homozygote, Sample B a heterozygote carrying short and long alleles, and Sample C a long-allele homozygote.

In this experiment, DNA extraction, PCR, electrophoresis, and sequence comparison were performed to analyze genetic differences among samples.

The DNA concentration of Sample A was 42.5 ng/µL, with an A260/A280 ratio of 1.82 and an A260/A230 ratio of 2.03.

In general, an A260/A280 ratio near 1.8 suggests relatively little protein contamination in a DNA sample. The A260/A230 ratio was also near 2.0, suggesting that contamination by salts or organic compounds was not substantial.

However, absorbance ratios alone cannot determine DNA integrity. Even degraded DNA can show apparently normal concentration and absorbance ratios, so the electrophoresis image should also be evaluated.

After PCR, electrophoresis showed bands of approximately 520 bp in Samples A and B. This indicates that the target region between the primers was present in both samples and that PCR amplification occurred.

The positive control also showed a band of approximately 520 bp, indicating that the PCR reagents, temperature conditions, electrophoresis, and staining steps generally functioned properly.

No main band of approximately 520 bp was observed in the negative control, suggesting that there was no major contamination of the reagents or work environment with target DNA.

However, a faint band was observed near 80 bp in the negative control. This was most likely a primer dimer produced by amplification after primers annealed to each other.

If primer dimers are strong, possible causes include excessive primer concentration, an annealing temperature that is too low, or complementarity between the primer sequences.

Sample A showed a weak band at approximately 250 bp in addition to the main band at approximately 520 bp. This may represent a nonspecific amplification product produced when the primers bound outside the target region.

Possible ways to reduce nonspecific bands include increasing the annealing temperature, adjusting primer or magnesium concentration, and reducing the number of cycles.

Sample C showed a band at approximately 700 bp, longer than the approximately 520 bp bands in Samples A and B.

This difference may indicate the presence of an insertion of approximately 180 bp between the primer-binding sites or a difference in the number of repeat sequences.

If Sample A shows only 520 bp, Sample B shows both 520 bp and 700 bp, and Sample C shows only 700 bp, Sample A can be estimated as a short-allele homozygote, Sample B as a heterozygote, and Sample C as a long-allele homozygote.

However, even when only one band is visible, allele dropout may have occurred, in which only one allele was amplified.

Therefore, confirming the genotype with replicate measurements, another primer pair, or sequence analysis is desirable.

Using the migration distance of the DNA ladder and the common logarithm of fragment size, the main band of Sample A was estimated at approximately 507 bp.

Because visual inspection suggested approximately 520 bp, the calculated and visual estimates were broadly consistent. The difference may have arisen from measurement of the band center, gel distortion, ladder migration distances, or approximation in the regression equation.

Sequence analysis showed that 468 of 472 bases matched the reference sequence, giving a sequence identity of 99.15%.

The high sequence identity suggests that the Sample A amplicon originated from the same gene region as the reference sequence.

There were four mismatching bases: three single-base substitutions and one single-base deletion.

To determine whether a single-base substitution represents a true genetic variant or a sequencing error, it is necessary to examine trace quality, the reverse-direction sequence, and replicate analyses.

Sequence-reading quality often decreases near sequence ends, which can lead to incorrect base calls. Low-quality regions should therefore be excluded before calculating sequence identity.

If a single-base insertion or deletion occurs within a protein-coding region, it may cause a frameshift that substantially alters the downstream amino-acid sequence.

By contrast, an insertion or deletion in multiples of three bases may add or remove amino acids while preserving the reading frame.

Base substitutions can also be synonymous substitutions that encode the same amino acid, missense substitutions that change an amino acid, or nonsense substitutions that create a stop codon.

Therefore, evaluating the effect of a mutation requires checking not only the DNA sequence difference but also changes in codons and the amino-acid sequence.

Even when sequence identity between samples is high, a short region alone may not distinguish closely related species or lineages.

To improve the reliability of species identification or phylogenetic analysis, multiple gene regions should be analyzed and compared with reliable sequences in public databases.

In this analysis, DNA concentration, purity, positive and negative controls, band size, and sequence identity were evaluated together.

In genetic analysis, conclusions should not be based solely on obtaining a target band. Nonspecific amplification, contamination, sequence quality, reference-sequence selection, and reproducibility must also be examined.

Overall, Samples A and B were estimated to contain the approximately 520 bp short region, whereas Sample C contained the approximately 700 bp long region.

The sequence of Sample A was 99.15% identical to the reference sequence, with a small number of base substitutions and a deletion.

These results indicate differences among the samples in both DNA fragment length and base sequence, which may be useful for genotype determination and comparisons among lineages.

Possible causes of failure to obtain the target band include insufficient DNA concentration, DNA degradation, contamination with PCR inhibitors, unsuitable primers, inappropriate annealing temperature, or reagent-addition errors. The positive-control result should be checked, and DNA quality and PCR conditions should be reevaluated.

If a band of the target size is observed in the negative control, contamination from reagents, pipettes, the workbench, or previously amplified DNA is suspected. In this case, positive sample results are also unreliable, so the work environment should be cleaned and the experiment repeated with fresh reagents.

Possible causes of smearing on the gel include DNA degradation, excessive template DNA, nonspecific amplification, excessive voltage, or salt contamination in the sample. DNA amount, PCR conditions, purification status, and electrophoresis conditions should be reviewed.

If sequence identity is low, possible causes include amplification of a different gene region, selection of an inappropriate reference sequence, incorrect sequence orientation, inclusion of low-quality regions, or alignment shifts caused by insertions or deletions. The reverse-complement sequence and quality scores should be checked and the sequences realigned.

Multiple bands may result from different-length alleles in a heterozygote, nonspecific amplification, multicopy genes, or a mixed sample. Band sizes, intensities, positive controls, and sequence data should be examined to determine the cause.