BCMB2001 Chap.14 PCR, Genotyping and Gel Electrophoresis
PCR, Genotyping and Gel Electrophoresis
PCR is controlled DNA synthesis. A template defines what can be copied, two primers define the boundaries and orientation of the target, a thermostable polymerase extends from primer ends, deoxyribonucleotides supply building blocks, buffer and ions support enzyme chemistry, and temperature cycling repeatedly separates strands, permits primer binding and supports extension.
A missing component has a mechanistic signature: no template means no target to copy; no primer means no defined start on that side; no polymerase means no extension; and a no-template control that produces a target-sized band indicates contamination or non-specific amplification rather than a clean negative.
Genotyping converts amplified length or sequence differences into allele calls.
A VNTR allele's repeat copy number changes amplicon length when primers flank the repeat. One band can be consistent with a homozygous locus, while two distinct allele-sized bands can indicate heterozygosity, provided controls and resolution are adequate. Agarose gel electrophoresis separates DNA chiefly by size under defined conditions because DNA migrates in an electric field through a porous matrix.
Topology complicates plasmids: supercoiled, linear and relaxed circular forms of the same base-pair length do not necessarily migrate together. DNA purity ratios at 260/280 and 260/230 provide evidence about contaminating classes, not a complete declaration of sample fitness. The chapter also connects reverse transcription, RT-PCR and Sanger sequencing to polymerase logic.
For restriction maps, fragment lengths are distances between cut sites. Linear molecules have endpoint fragments; circular molecules include the wrap-around distance and fragment lengths must sum to the total molecule length. Practice maps use new cut positions and explicit AskSia weighting rather than inheriting an unstated university mark budget.
What this chapter covers
- 01
Buccal-cell DNA isolation and the trade-off between rapid preparation, purity and downstream suitability
- 02
PCR template, primers, polymerase, nucleotides, buffer, ions and temperature-cycle logic
- 03
Positive and no-template controls as evidence separating biological absence from technical failure or contamination
- 04
Reverse transcription, RT-PCR and Sanger dideoxy termination as extensions of template-directed synthesis
- 05
Amplicon size, VNTR repeat number, homozygous and heterozygous genotype interpretation
- 06
Agarose separation, DNA purity ratios, plasmid conformation and restriction-fragment arithmetic
Predict circular restriction fragments
- +1 (AskSia)Place the sites in circular order: 0, 2000, 2900, then return to 3600, which is the same position as 0 on the circle.
- +1 (AskSia)The first interval is 2000 minus 0, giving a 2000-base-pair fragment.
- +1 (AskSia)The second interval is 2900 minus 2000, giving 900 base pairs; the wrap-around interval is 3600 minus 2900, giving 700 base pairs.
- +1 (AskSia)The predicted set is 2000, 900 and 700 base pairs. Their sum is 3600 base pairs, conserving the full plasmid length.
Key terms
- Primer
- A short nucleic-acid strand that base-pairs with a template and supplies an extendable end for polymerase.
- No-template control
- A PCR condition containing reaction components but no intended template, used to detect contamination or reagent-derived amplification.
- Amplicon
- The DNA product amplified between the primer-defined boundaries of a PCR reaction.
- VNTR
- A locus containing a variable number of tandem repeat units, producing allele-length differences when flanking regions are amplified.
- Dideoxy nucleotide
- A nucleotide analogue lacking the chemical group required for continued chain extension, used to terminate Sanger products.
- Restriction fragment
- A DNA segment bounded by restriction cuts or by a cut and a physical end on a linear molecule.
PCR, Genotyping and Gel Electrophoresis FAQ
What does a clean negative PCR result prove?
Only what the controls support. If the positive control amplifies and the no-template control remains clean, absence of a sample band supports absence, poor primer match or target below detection rather than global reaction failure or contamination. If the positive control fails, the sample negative is uninterpretable. Read controls before genotype lanes.
Why are two primers required?
They bind opposite template strands with orientations that point extension into the target region. Together they define both boundaries and enable exponential enrichment of the bounded product. A single primer can support extension but does not create the same two-ended amplification cycle. Primer sequence and orientation therefore determine specificity and product length.
How does VNTR length become genotype?
Flanking primers hold constant while the number of internal repeat units varies, so allele length equals the fixed flanks plus repeat-unit length multiplied by copy number. One resolved allele size can indicate homozygosity; two sizes can indicate heterozygosity. Controls, gel resolution and alternative amplification artefacts must still be considered before making the call.
Why can plasmid forms run at unexpected positions?
Gel migration depends on conformation as well as base-pair length. A compact supercoiled plasmid can move differently from a linear molecule of the same length, while a relaxed or nicked circle experiences another effective shape. Use a restriction digest that produces a known linear form when you need length comparison, and do not assign size from untreated plasmid position alone.
What do DNA purity ratios tell me?
Absorbance ratios compare the nucleic-acid signal region with wavelengths where protein or other contaminants contribute. Values far from the expected clean-sample pattern suggest contamination, but acceptable ratios do not guarantee intact, amplifiable DNA. Interpret ratios alongside concentration, gel behaviour and a functional PCR control.
How is Sanger sequencing related to PCR?
Both use template-directed polymerase extension, primers and nucleotide substrates. Sanger chemistry adds labelled chain-terminating dideoxy analogues so products end at different sequence positions and can be ordered by size. It reads the sequence of extension products rather than simply accumulating one bounded product for endpoint detection.
Exam move
Draw a PCR tube and assign every component one job and one failure prediction. Then sketch three cycles with primer orientation so exponential amplification is derived rather than memorised. For gels, read controls first, ladder second and samples last; write the expected band set before looking at the observed lanes.
Practise VNTR arithmetic with a fixed flank term and a repeat term, checking that inferred copy number is physically sensible. On restriction maps, keep linear and circular algorithms separate and require fragment lengths to conserve total DNA. Add a plasmid-conformation warning beside every uncut lane. Finally, interpret purity ratios as evidence of possible contamination rather than as an automatic pass or fail.
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