Reading PCR, sequencing, and DNA blot results

MCAT content library / Biology & biochemistry / Reading PCR, sequencing, and DNA blot results

Choose the DNA method that answers the question, then read the signal without claiming more than the assay can establish.

About 17 minutes, including the worked example and checks.

Start with the measurement target

The MCAT can place PCR, gel electrophoresis, hybridization, Southern blotting, or DNA sequencing inside an experiment and ask what a result means. The fastest route is not to memorize a list of techniques. Ask what physical feature the assay reads: amplification of a bounded DNA region, migration of DNA fragments by size, hybridization to a complementary sequence, or nucleotide order itself.¹

That distinction sets the ceiling on the conclusion. A band at an expected size can support the presence of a product of that approximate length under the assay conditions. It does not by itself establish the exact sequence. Conversely, a sequence read can identify a nucleotide substitution in the read region, but it does not automatically quantify how much DNA was present in the starting sample.

Concept: PCR primers define the amplicon

PCR repeatedly copies a selected DNA segment through cycles that include strand separation, primer annealing, and DNA synthesis. Two primers provide free 3′ hydroxyl groups for extension and define the amplified boundaries. Each primer is written 5′ to 3′, anneals antiparallel to a complementary template strand, and must point its extendable 3′ end into the region to be copied.²³

When a question prints one reference strand 5′ to 3′, the forward primer commonly matches the left-hand sequence of that displayed strand because it anneals to the opposite strand. The reverse primer is the reverse complement of the displayed right-hand boundary. Do not merely choose two sequences that are complementary somewhere in the locus; their orientation must permit synthesis toward one another.

The final PCR product includes the primer-defined endpoints. If an insertion lies between unchanged primer sites, the insertion increases amplicon length. If a primer-binding site changes, amplification efficiency can fall even when the rest of the locus is still present. A no-template control tests for reagent contamination, while a separate positive amplification control can help show that amplifiable DNA and the reaction system were present.

Pattern: read a gel as size evidence

DNA carries a negatively charged phosphate backbone and migrates toward the positive electrode during electrophoresis. In an agarose matrix, smaller DNA fragments generally move farther than larger fragments under the same run conditions. A size ladder provides reference fragments; the band position is therefore interpreted relative to the ladder rather than as an intrinsic “brightness equals size” rule.

Band intensity can vary with DNA amount, staining, loading, imaging, saturation, and fragment properties. Unless the experiment establishes a quantitative relationship and appropriate controls, use a gel primarily for presence and approximate size. One sample can also contain several fragments that comigrate or one fragment below the detection threshold, so an apparently simple lane is not a complete sequence description.

A Southern blot adds sequence-specific hybridization to the size separation. DNA fragments are separated, transferred to a membrane, and exposed to a labeled probe complementary to a target sequence. A detected band therefore combines two kinds of evidence: a fragment migrated to that approximate size and contained sequence sufficiently complementary to the probe under the hybridization conditions.⁵

Worked application: one locus, three DNA readouts

Researchers study an original fictional locus, mrx. A reference allele produces a 420-base-pair PCR product using primers F and R. Variant I contains a 300-base-pair insertion between unchanged primer sites, so its expected PCR product is 720 bp. Variant P keeps the reference internal sequence but changes the last nucleotide of the genomic site paired with the 3′ end of primer F. The exercise stipulates that this mismatch prevents detectable amplification with primer pair F/R under the chosen conditions.

A second primer pair amplifies an unrelated 250-bp control locus from all three purified DNA samples. A restriction digest used for Southern analysis cuts outside the mrx target and produces a 2.4-kb reference fragment; the 300-bp insertion makes that fragment 2.7 kb. The probe lies inside mrx but outside the F primer-binding site, so the single primer-site change in P does not alter probe complementarity. Assume complete digestion, one relevant fragment per genome copy, equal sample loading, and adequate resolution.

Sample B is the straightforward length case: both PCR and Southern analysis shift by 300 bp because the insertion lies inside both measured intervals. Sample C is different. The control PCR shows that the sample contains amplifiable DNA under the run conditions, and the Southern probe still detects the expected 2.4-kb target-containing fragment. The missing F/R band therefore does not justify “the gene is absent.” Under the stipulated model, a primer-site problem explains the discordant result.

If the researchers sequence the relevant primer-binding region in C and obtain the stated single-base change, that result directly addresses the nucleotide-level explanation. Sequencing is valuable here because DNA sequencing determines nucleotide order rather than only fragment size or probe binding.⁴

Rule: a negative band is not a deletion

Failure to detect a PCR product is an observation about that amplification reaction. Possible explanations can include absent target DNA, changed primer-binding sequence, degraded or insufficient template, inhibitors, unsuitable reaction conditions, or a technical failure. The experiment narrows that list only when its controls distinguish among those possibilities.

This is a useful MCAT inference rule: absence of evidence from one readout is not automatically evidence of physical absence. Ask which step must succeed before the signal can appear. A Southern signal or a second primer pair can show that target-region DNA is present even when the original primer pair fails. Conversely, a clean PCR band does not prove that every base in the amplicon matches the reference sequence.

Choose the assay that can distinguish the models

• PCR + gel: useful when a bounded target must be amplified and product presence or size can separate the hypotheses.
• Southern blot: useful when a complementary probe should identify a target-containing restriction fragment and its approximate size.
• DNA sequencing: useful when the competing models differ by nucleotide order within the readable region.
• Another control or primer pair: useful when the main uncertainty is whether a negative result reflects the sample, the target site, or the assay.

Method choice should follow the competing explanations. If two variants differ by a single nucleotide but produce fragments of the same length, an ordinary size gel may not separate them. If one model deletes the probe-binding sequence while another only changes a distant primer site, hybridization can be informative. The most sophisticated technique is not automatically the best answer; the best answer is the one whose possible outcomes differ between the models being tested.

Check your reasoning

Choose an answer before opening its feedback. Each option explains the reasoning behind that choice.

Continue learning

• Cloning and recombinant DNA
• Measuring Gene Expression and Testing Gene Function
• Experimental Controls: What a Comparison Can Show
• Browse Biology & Biochemistry MCAT lessons

Sources

1. AAMC. What’s on the MCAT Exam? Content Outline. See Biological and Biochemical Foundations, content category 1B.
2. National Human Genome Research Institute. Polymerase Chain Reaction (PCR) Fact Sheet.
3. National Human Genome Research Institute. Primer.
4. National Human Genome Research Institute. DNA Sequencing.
5. NCBI Bookshelf. Biochemistry, Replication and Transcription. Southern blotting, PCR, and sequencing overview.