DNA sequencing and DNA profiling (HSC Biology Module 5)
“Investigate the use of technologies to determine inheritance patterns in a population using, for example, but not limited to: DNA sequencing and profiling”
DNA sequencing reads the exact order of bases (A, T, G, C), classically by the Sanger chain-termination method (labelled ddNTPs stop synthesis, fragments are size-separated, and the terminal base is read in order), with next-generation sequencing doing millions of reads in parallel. DNA profiling instead produces a pattern of fragment lengths: PCR amplifies variable short tandem repeats, then gel electrophoresis separates the fragments by size (DNA runs to the positive electrode, shorter fragments travel further) into a banding pattern. Sequencing detects a specific allele or mutation; profiling identifies or relates individuals. To read a gel, line the sample bands up against a labelled size ladder and compare lanes. Applications span forensics, paternity, conservation/population studies and tracking disease alleles.
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What this dot point is asking
NESA wants you to investigate the technologies scientists use to determine inheritance patterns in a population, using DNA sequencing and DNA profiling as the named examples. You must be able to describe how each technology works, clearly distinguish what each one produces (sequencing gives the order of bases; profiling gives a pattern of fragment lengths), and apply them to real situations such as forensics, paternity, conservation and tracking a disease allele.
A common exam task is to "read" a gel or a DNA profile - interpret a banding pattern to decide whether two samples match, or which parent passed on which allele. The command words range from "outline" and "describe" up to "compare" and "evaluate", so match your depth to the verb.
The answer
Two different technologies answer two different questions about DNA. Sequencing asks "what is the exact order of the bases?" Profiling asks "what does this person's pattern of variable regions look like, and does it match another sample?" Keep that contrast at the front of your mind - most marks on this dot point come from getting it right.
DNA sequencing: reading the order of bases
DNA sequencing determines the precise order of the nucleotide bases (A, T, G, C) along a stretch of DNA.
The Sanger (chain-termination) method is the classic technique:
- A reaction mix contains the single-stranded template DNA, a primer, DNA polymerase, the four normal nucleotides (dNTPs), and a small proportion of chain-terminating dideoxynucleotides (ddNTPs) - one for each base, each tagged with a different fluorescent colour.
- DNA polymerase builds new complementary strands. Most of the time it adds a normal nucleotide, but occasionally it adds a ddNTP, which stops synthesis because a ddNTP lacks the 3' -OH group needed to bond to the next nucleotide.
- Because termination happens at every position by chance across the many copies, the result is a set of fragments of every possible length, each ending in a labelled base.
- The fragments are separated by size (shortest to longest, one base at a time) by capillary electrophoresis. As each fragment passes a detector, the colour of its final base is read, and the order of colours spells out the sequence, 5' to 3'.
Next-generation (high-throughput) sequencing - in brief - does not read one fragment at a time. Instead it sequences millions of short DNA fragments in parallel, then a computer overlaps the reads to reconstruct the full sequence. It is vastly faster and cheaper per base, which is why whole genomes can now be sequenced routinely.
Reading a sequence: a short electropherogram trace is just a row of coloured peaks; reading the peaks left to right gives the base order, e.g. a trace of green-red-black-blue reads as A - T - G - C (the exact colour code depends on the machine).
DNA profiling: a pattern of fragment lengths
DNA profiling (also called DNA fingerprinting) does not read the bases. It produces a pattern of fragment lengths from highly variable regions of the genome - typically short tandem repeats (STRs).
- Short tandem repeats (STRs) are short DNA sequences (for example, the four bases
GATA) repeated a variable number of times in a row. The number of repeats differs between individuals, so the length of the region differs. A person has two alleles at each STR locus (one from each parent). - PCR (polymerase chain reaction) amplifies the chosen STR regions, making millions of copies from even a tiny sample (a hair root, a spot of blood, a cheek swab) so the fragments can be detected.
- Gel electrophoresis separates the amplified fragments by size. The DNA is loaded into wells at one end of a gel; an electric field is applied; because DNA is negatively charged it migrates towards the positive electrode, and the gel sieves the fragments so shorter fragments travel further. The result is a set of bands - a DNA profile.
Using several STR loci together, the combined banding pattern becomes effectively unique to an individual (apart from identical twins).
Reading the gel above
Read down each lane against the ladder: a band level with the ladder's 300 bp line is a 300 bp fragment. The crime-scene sample has bands at 300 and 100 bp. Scanning across, suspect 2 has exactly the same two bands, while suspect 1 (400 + 200) and the victim (400 + 100) do not match. So the crime-scene DNA matches suspect 2. Real casework uses many more loci, but the reading logic is identical: line the bands up against the ladder and compare lanes.
How profiling reveals inheritance patterns
Because a person inherits one STR allele from each parent, band patterns follow Mendelian inheritance. A child's bands must each be found in one of the biological parents. This is the basis of paternity testing (does the child's non-maternal band appear in the alleged father?) and of building pedigrees in families or wild populations. Across a whole population, comparing profiles measures genetic diversity and relatedness, which is how conservation biologists detect inbreeding in endangered species.
Applications
- Forensics. Match crime-scene DNA to a suspect (or exclude an innocent person) by comparing STR band patterns.
- Paternity / parentage. Check that a child's alleles can be accounted for by the alleged parents.
- Conservation and population studies. Measure genetic diversity, relatedness and inbreeding in endangered populations; identify individuals from non-invasive samples (hair, scat).
- Disease-allele tracking. Sequencing detects the exact disease-causing mutation; profiling can track a linked STR marker through a family pedigree to follow how a disease allele is inherited.
Sequencing reads the order of bases; profiling reads a pattern of fragment lengths. DNA sequencing (Sanger chain-termination, or fast parallel next-generation sequencing) gives the exact A-T-G-C order, so it can find a single-base mutation. DNA profiling uses PCR to amplify variable STR regions, then gel electrophoresis to separate the fragments by size (DNA is negative, so it runs to the + electrode and shorter fragments travel further), producing a banding pattern used to identify or relate individuals.
Worked example 1 - distinguish the two technologies (2 marks)
Question. Distinguish between DNA sequencing and DNA profiling.
Model answer. DNA sequencing determines the exact order of nucleotide bases (A, T, G, C) along a length of DNA. DNA profiling instead produces a pattern of fragment lengths from variable regions such as short tandem repeats, identifying or matching individuals without reading the individual bases.
Marker's note: 1 mark for the sequencing definition (order of bases), 1 mark for the profiling definition (pattern of fragment lengths). The contrast must be explicit; describing one only, or blurring the two, caps at 1 mark.
Worked example 2 - read the gel (4 marks)
Question. A size ladder shows bands at 100, 200, 300 and 400 bp. A child shows bands at 200 and 300 bp; the mother shows 200 and 400 bp; the alleged father shows 100 and 300 bp. Could the alleged father be the biological father?
Model answer. At one STR locus a person carries two alleles, one from each biological parent, seen as two bands. The child's bands are 200 and 300 bp. The mother can supply the 200 bp allele, so the child's 300 bp allele must have come from the biological father. The alleged father carries a 300 bp band, so he could have contributed it; he therefore cannot be excluded as the biological father at this locus. (A definitive test would examine many loci, since a single matching locus is not conclusive.)
Marker's note: 1 mark for the one-allele-per-parent principle, 1 mark for assigning the maternal allele, 1 mark for showing the alleged father carries the required paternal band, 1 mark for the correct, appropriately cautious conclusion.
Worked example 3 - describe Sanger sequencing (5 marks)
Question. Describe the Sanger (chain-termination) method and explain how the products are used to read the base sequence.
Model answer. A reaction contains the template DNA, a primer, DNA polymerase, the four nucleotides, and a small proportion of fluorescently labelled chain-terminating dideoxynucleotides (ddNTPs). DNA polymerase extends new strands; whenever a ddNTP is incorporated, synthesis stops because the ddNTP lacks the 3' -OH group needed to add the next base. Termination at every position produces fragments of every possible length, each ending in a labelled base. The fragments are separated by size (capillary electrophoresis) from shortest to longest, and as each passes a detector its terminal colour is read, so the order of colours gives the base sequence 5' to 3'.
Marker's note: 1 mark each for the components, the role of the ddNTP (missing 3' -OH causing termination), the set of different-length fragments, the size separation, and reading the terminal base in order. Band 6 answers tie the missing 3' -OH directly to termination.
- Confusing sequencing with profiling
- Sequencing reads the order of bases; profiling reads a pattern of fragment lengths. Swapping these is the single most penalised error on this dot point.
- Getting gel migration backwards
- DNA is negatively charged, so it runs towards the positive electrode, and shorter fragments travel further (not the other way round). Larger fragments stay near the wells.
- Forgetting PCR's job
- PCR amplifies the DNA so there is enough to detect - it does not "cut" or "separate" the DNA. Separation is the gel's job; the variability comes from the STRs.
- Treating one matching band as proof
- A match at a single locus is consistent with, but does not prove, identity or paternity. Reliability comes from combining many independent STR loci.
- Lead every answer with the core contrast
- "Sequencing = order of bases; profiling = pattern of fragment lengths." Markers reward candidates who keep the two technologies clearly separate.
- For a "read the gel" item, work against the ladder
- Line each sample band up with the labelled ladder band of known size, then compare lanes for a match. State the sizes you read - don't just say "they look the same".
- Match depth to the command word
- "Outline" wants brief points; "describe" wants the steps in order; "compare" wants the same features for both technologies; "evaluate" wants a justified judgement weighing strengths and limitations.
- Name the tech precisely
- PCR (amplify), STR (the variable region), gel electrophoresis (separate by size), Sanger / ddNTP (sequencing), next-generation sequencing (parallel, fast, cheap). Precise terms lift a Band 4 answer to Band 6.
Imagine DNA as an enormous book written in only four letters. Sequencing is reading the book letter by letter to find the exact spelling - perfect for spotting a single typo, like a disease-causing mutation. Profiling doesn't read the words at all; it's more like measuring the lengths of a few special paragraphs that everyone has but at different lengths. To do that, scientists first photocopy those paragraphs millions of times (that's PCR), then run the pieces through a jelly with electricity (gel electrophoresis): the small pieces zip further, the big pieces lag, so you get a pattern of stripes, like a supermarket barcode. Because you get half your "barcode" from each parent, matching stripes can show who a sample came from or who a child's parents are. That barcode idea is what solves crimes, settles paternity, and helps zookeepers keep endangered animals from becoming too inbred.
Practice questions
Original practice questions graded from foundation to exam level, each with a full worked solution. Try them before revealing the solution.
foundation2 marksDistinguish between DNA sequencing and DNA profiling.Show worked solution →
1 mark - DNA sequencing. DNA sequencing determines the exact order of nucleotide bases (A, T, G, C) along a length of DNA.
1 mark - DNA profiling. DNA profiling produces a pattern of fragment lengths (a banding pattern) from variable regions such as short tandem repeats, without reading the individual bases.
The mark hinges on the contrast: sequencing reads the order of bases, profiling compares the size/number of repeats. An answer that treats the two as the same idea earns at most 1 mark.
foundation3 marksOutline the role of each of the following in producing a DNA profile: (a) PCR, (b) short tandem repeats (STRs), (c) gel electrophoresis.Show worked solution →
- 1 mark - PCR
- The polymerase chain reaction amplifies the target DNA, making millions of copies of the chosen STR regions so there is enough DNA to detect.
- 1 mark - STRs
- Short tandem repeats are short DNA sequences repeated a variable number of times; the number of repeats differs between individuals, so they are the variable feature the profile is built from.
- 1 mark - gel electrophoresis
- Gel electrophoresis separates the amplified fragments by size: an electric field pulls the negatively charged DNA through the gel, and shorter fragments travel further, producing a banding pattern.
Each component must be tied to its specific job (amplify, vary, separate); naming the technique without its function does not earn the mark.
foundation2 marksExplain why DNA fragments separate by size during gel electrophoresis.Show worked solution →
1 mark - the driving force. DNA carries a negative charge (from its phosphate groups), so when an electric field is applied it migrates through the gel towards the positive electrode (anode).
1 mark - the size effect. The gel acts as a molecular sieve: shorter fragments move faster and travel further, while longer fragments are held back, so fragments sort into bands by length.
Both the charge/direction and the size relationship are needed. Saying fragments "just spread out" without the charge or the size mechanism caps at 1 mark.
core4 marksA laboratory analyses one STR locus. A fragment-length ladder shows reference bands at 100, 200, 300 and 400 base pairs. At this locus a child shows bands at 200 bp and 300 bp; the mother shows 200 bp and 400 bp; the alleged father shows 100 bp and 300 bp. Determine whether the alleged father could be the biological father, justifying your answer using the inheritance of alleles.Show worked solution →
- 1 mark - principle
- At one STR locus a person carries two alleles, one inherited from each biological parent; the two are seen as two bands (unless the person is homozygous and shows one).
- 1 mark - assign the maternal allele
- The child's bands are 200 bp and 300 bp. The mother (200, 400) can supply the 200 bp allele, so the child's 300 bp allele must have come from the biological father.
- 1 mark - test the alleged father
- The alleged father's bands are 100 bp and 300 bp. He carries a 300 bp allele, so he could have contributed the child's paternally derived 300 bp band.
- 1 mark - conclusion
- The alleged father cannot be excluded at this locus - he could be the biological father. (A full paternity test would examine many loci; matching at one locus alone is not conclusive proof.)
Top responses note both that the match is consistent AND that one locus is not definitive - that nuance separates a complete answer from a partial one.
core4 marksCompare DNA sequencing and DNA profiling in terms of the information each produces and a situation in which each is the more appropriate technology.Show worked solution →
Award up to 4 marks for a genuine comparison (both similarities/differences) plus appropriate applications.
Information produced (2 marks). Sequencing reads the precise order of every base in a region, so it can detect a single-base change such as a point mutation that causes a disease allele. Profiling produces only a pattern of fragment lengths from variable repeat regions; it identifies or relates individuals but does not reveal the base sequence of a gene.
Appropriate situations (2 marks). Sequencing is appropriate when the goal is to identify a specific allele or mutation - for example confirming a cystic fibrosis allele or tracking a disease allele through a population. Profiling is appropriate when the goal is to identify or match individuals - for example forensic identification, paternity testing or measuring genetic diversity in a conservation population.
A response that only describes one technology, or gives applications without the underlying reason, does not reach full marks. The discriminator is "order of bases" (sequencing) versus "pattern of fragment lengths" (profiling).
core5 marksDescribe the Sanger (chain-termination) method of DNA sequencing, and explain how the products are used to read the base sequence.Show worked solution →
- 1 mark - components
- A sequencing reaction contains the single-stranded DNA template, a primer, DNA polymerase, the four normal nucleotides (dNTPs), and a small proportion of chain-terminating dideoxynucleotides (ddNTPs), each base labelled with a different fluorescent colour.
- 1 mark - chain termination
- DNA polymerase extends new strands along the template. Whenever a ddNTP is incorporated instead of a normal nucleotide, synthesis stops, because the ddNTP lacks the 3' -OH group needed to add the next nucleotide.
- 1 mark - a set of fragments
- Termination happens at every position by chance across the many copies, producing a set of fragments of every possible length, each ending in a labelled ddNTP that marks its final base.
- 1 mark - separation
- The fragments are separated by size (by capillary or gel electrophoresis), from shortest to longest, differing by one nucleotide.
- 1 mark - reading the sequence
- As fragments pass a detector in size order, the colour of the terminal base at each step is read off, and the order of colours gives the base sequence 5' to 3'.
Band 6 answers explicitly link the missing 3' -OH to chain termination, and the order of fragment sizes to reading bases one at a time.
exam7 marksA national wildlife agency wants to manage a small, isolated population of an endangered marsupial and is choosing between DNA sequencing and DNA profiling. Evaluate the use of these two technologies for determining inheritance patterns and genetic health in this population.Show worked solution →
"Evaluate" requires a judgement that weighs the strengths and limitations of each technology against the agency's purpose. A Band 6 response reaches a reasoned conclusion, not just a description.
- What each technology offers (2-3 marks)
- DNA profiling (PCR of STR loci, then electrophoresis) is fast, cheap and needs only tiny samples (hair, scat, a blood spot). It produces banding patterns that let the agency identify individuals, establish parentage, build pedigrees and measure genetic diversity / heterozygosity across the population - directly addressing inheritance patterns and inbreeding risk. DNA sequencing reads the actual base order, so it can detect specific deleterious alleles or mutations and reveal fine-scale relatedness, but it is more expensive and data-heavy.
- Strengths and limitations weighed (2-3 marks)
- Profiling is excellent for population-scale screening and relatedness but cannot tell you which alleles are present or whether a harmful mutation is spreading. Sequencing answers those allele-level questions but is costlier per animal and may be unnecessary for routine pedigree work. Sample quality, cost per animal and the agency's specific question all bear on the choice.
- Judgement (1-2 marks)
- A supported conclusion: DNA profiling is the more appropriate primary tool for routine monitoring of inheritance patterns and genetic diversity (cheap, scalable, identifies individuals and relatedness), with targeted DNA sequencing reserved for confirming specific disease alleles or investigating particular loci of concern. An answer that lists features of each without an explicit, justified judgement caps below full marks.
exam6 marksExplain how DNA profiling can be used to track the inheritance of a disease-causing allele through several generations of a family, and discuss one limitation of using a standard STR profile for this purpose.Show worked solution →
Target a sequenced response that links profiling, linkage and pedigree tracking, then identifies a genuine limitation.
- How a profile tracks an allele (2-3 marks)
- A DNA profile reveals the STR alleles (band sizes) each family member carries at chosen loci. If an STR marker lies physically close (linked) to the disease gene on the same chromosome, the marker allele tends to be inherited together with the disease allele. By profiling several generations and comparing band patterns with the pedigree of affected and unaffected individuals, the marker allele co-segregating with the disorder can be tracked, predicting who carries the disease allele.
- Why this works across generations (1-2 marks)
- Each individual inherits one allele from each parent, so band patterns follow Mendelian inheritance; matching bands to affected status across a pedigree shows the inheritance pattern of the linked marker (and thus the allele) through the family.
- Limitation (1-2 marks)
- A standard STR profile does not read the disease gene itself - it relies on linkage, so recombination (crossing over) between the marker and the gene can break the association and give a false prediction. (Accept also: the profile shows fragment lengths, not the base change, so it cannot confirm the exact mutation - direct sequencing would be needed for that.)
Full marks need the linkage mechanism, the across-generation inheritance logic, AND a valid limitation that follows from profiling reading lengths rather than bases.
Practise this
Sources & how we know this
- Biology Stage 6 Syllabus (2017) — NESA
- Biology HSC exam papers — NESA