dna mod lec 3

What is a VNTR?

A VNTR stands for Variable Number Tandem Repeat. It is a region of DNA where a short sequence is repeated multiple times in a row. Different people can have different numbers of repeats, so the length of that DNA region can vary between people.

Why are VNTRs useful in forensic DNA analysis?

VNTRs are useful because different people often have different repeat numbers. This means they can create a DNA banding pattern that helps identify or exclude a person. They were especially important in early DNA fingerprinting.

How does VNTR analysis basically work?

DNA is extracted, then cut into fragments using restriction enzymes. The fragments are separated by size using gel electrophoresis. The VNTR regions are then visualised using a labelled probe. The final banding pattern can be compared between samples.

What does a restriction enzyme do in VNTR analysis?

A restriction enzyme cuts DNA at specific DNA sequences. This creates DNA fragments of different lengths. If people have different numbers of VNTR repeats, the fragments will be different sizes.

Why do VNTR fragments separate on a gel?

DNA fragments separate by size during gel electrophoresis. Smaller fragments move further through the gel, while larger fragments move more slowly and stay closer to the top.

What is a probe in VNTR analysis?

A probe is a labelled piece of DNA that binds to a specific target sequence. In VNTR analysis, the probe binds to the VNTR region so that the bands can be detected.

What is hybridisation in VNTR analysis?

Hybridisation is when the labelled DNA probe binds to its matching DNA sequence. This lets scientists see where the VNTR fragments are on the membrane or gel.

Why did VNTR analysis take longer than modern STR analysis?

VNTR analysis was slower because it needed larger DNA fragments, restriction enzyme cutting, gel electrophoresis, transfer to a membrane, probe binding, and visualisation. This process could take days.

What are the main limitations of VNTR analysis?

VNTR analysis needs relatively large amounts of high-quality DNA. It is more time-consuming than STR analysis, and it is harder to use if the DNA is degraded because degraded DNA breaks into smaller fragments.

Why is degraded DNA a problem for VNTR analysis?

VNTR analysis targets large DNA fragments. If DNA is degraded, it has already broken into smaller pieces, so the large VNTR regions may not survive well enough to analyse.

What is the simple difference between VNTRs and STRs?

VNTRs are longer repeated DNA regions, while STRs are shorter repeated DNA regions. STRs are easier to analyse because they need less DNA and work better with degraded DNA.

What does STR stand for?

STR stands for Short Tandem Repeat. It is a short DNA sequence, usually 2 to 5 base pairs long, that is repeated multiple times in a row.

Why are STRs useful in forensic DNA analysis?

STRs are useful because the number of repeats varies between people. By looking at several STR loci, scientists can create a DNA profile that is highly individualising.

How does STR analysis basically work?

DNA is extracted, specific STR regions are amplified using PCR, the amplified fragments are separated by size using electrophoresis, and the STR pattern is used to create a DNA profile.

Why is PCR important in STR analysis?

PCR is important because it copies specific DNA regions many times. This means scientists can analyse very small amounts of DNA.

Why is STR analysis faster than VNTR analysis?

STR analysis is faster because it uses PCR to amplify small target regions directly. It does not require cutting the whole genome into fragments and using Southern blot-style VNTR analysis.

Why does STR analysis work better with degraded DNA than VNTR analysis?

STRs are smaller DNA regions. Because degraded DNA is broken into small pieces, STR targets are more likely to still be intact compared with larger VNTR regions.

What does a locus mean in STR analysis?

A locus is a specific location on the DNA. In STR analysis, each locus is a specific STR region being examined.

Why do we usually analyse many STR loci instead of just one?

One STR locus is not enough to strongly identify someone because many people may share the same repeat number at one locus. Using many independent STR loci makes the chance of a random match much lower.

What is an allele in STR analysis?

An allele is the repeat number someone has at a particular STR locus. For example, if someone has 10 repeats at a locus, their allele may be called 10.

Why can someone have two STR alleles at one locus?

People usually have two copies of each autosomal chromosome, one from their mother and one from their father. So at one STR locus, they can inherit one allele from each parent.

What does it mean if someone has one peak/band at an STR locus?

One peak usually means the person is homozygous at that locus. This means they inherited the same repeat number from both parents, so both fragments are the same size and appear together.

What does it mean if someone has two peaks/bands at an STR locus?

Two peaks usually means the person is heterozygous at that locus. This means they inherited two different repeat numbers, one from each parent.

What is a homozygous STR result?

A homozygous STR result means both alleles at that locus are the same. For example, 10,10 means the person inherited 10 repeats from both parents.

What is a heterozygous STR result?

A heterozygous STR result means the two alleles at that locus are different. For example, 10,14 means the person inherited one allele with 10 repeats and one allele with 14 repeats.

Why do STR fragments of different sizes move differently during electrophoresis?

Fragments with fewer repeats are shorter, so they move further through the gel or capillary. Fragments with more repeats are longer, so they move more slowly.

What is the forensic significance of STRs?

STRs are the current gold standard in forensic DNA analysis. They are accurate, sensitive, relatively fast, and can be used with small or degraded DNA samples.

What is D1S80?

D1S80 is a VNTR marker on chromosome 1. It has a repeat number that varies between people, so it can be used for personal identification.

Why is D1S80 useful for personal identification?

D1S80 has many possible repeat numbers, so different people can have different fragment sizes. This variation can help distinguish between individuals.

What does it mean that D1S80 is intergenic?

Intergenic means it is located between genes, rather than inside a gene. This is useful because forensic DNA profiling usually targets variable non-coding regions rather than protein-coding genes.

What is CODIS?

CODIS stands for Combined DNA Index System. It is a DNA database system developed in the USA and managed by the FBI. It uses STR profiles to compare DNA samples.

Why are CODIS STR markers powerful?

CODIS uses multiple STR loci, mostly on different chromosomes. Because the markers are independent, combining them makes the overall DNA profile much more individualising.

Why is it useful that CODIS markers are mostly independent?

If markers are independent, the result at one locus does not strongly predict the result at another locus. This means the probabilities can be combined to make the chance of a random match much smaller.

What is amelogenin used for in forensic STR panels?

Amelogenin is used as a sex marker. It helps indicate whether the DNA profile is likely from a biological male or female.

Why was the CODIS panel expanded from 13 to 20 loci?

Adding more loci increases the power of discrimination. This means the chance of a random unrelated person having the same DNA profile becomes even lower.

What is match probability?

Match probability is the estimated chance that a random person from a population would have the same DNA profile. It helps explain how strong a DNA match is.

Why can we not say a DNA match is 100 percent certain?

A DNA match is interpreted statistically. Even if two profiles match, scientists report how rare that profile is in a population, rather than saying it is absolutely impossible for anyone else to match.

What information is needed to calculate match probability?

You need allele frequencies, genotype frequencies, and then you combine the genotype frequencies across loci using the product rule.

What is an allele frequency?

Allele frequency is how common a particular allele is in a population. For example, it tells us how common allele 15 is at a particular STR locus.

What is a genotype frequency?

Genotype frequency is how common a particular pair of alleles is in a population. For example, how common 15,15 or 16,17 is at a specific STR locus.

What is the product rule in DNA match probability?

The product rule means multiplying the genotype frequencies from different independent loci together. This gives the overall probability of the full DNA profile occurring randomly.

Why does adding more STR loci lower the match probability?

Each extra independent locus adds more information. When the probabilities from many loci are multiplied together, the overall chance of a random match becomes much smaller.

Why does population data matter in DNA interpretation?

Allele frequencies can differ between populations. If the wrong population data is used, the match probability may be less accurate.

Why might allele frequencies differ between populations?

Different populations can have different genetic histories, ancestry patterns, and allele distributions. This means an allele may be rare in one population but more common in another.

Why might you use more or different loci in DNA analysis?

More or different loci may be useful for degraded samples, mixed samples, closely related individuals, or populations where the usual loci do not give enough discrimination.

Why might degraded samples need more loci?

In degraded samples, some loci may fail because the DNA is broken. Testing more loci increases the chance of getting enough usable information for a partial profile.

Why might mixed samples need more loci?

Mixed samples contain DNA from more than one person. More loci can give more information and may help separate or interpret the contributors more clearly.

Why are closely related individuals harder to distinguish by DNA profiling?

Closely related people share more alleles than unrelated people. This means their STR profiles are more similar, so more loci may be needed to improve discrimination.

What is multiplexing in STR analysis?

Multiplexing means amplifying multiple STR loci in the same PCR reaction. This saves time, uses less sample, and reduces the need for many separate tests.

Why is multiplexing useful?

Multiplexing is useful because forensic samples may be very small. Instead of using lots of DNA and doing many separate PCR reactions, many STR markers can be amplified at once.

Why can multiplexing be technically difficult?

Multiplexing is difficult because many primer pairs are used in one reaction. The primers must not interfere with each other or bind to the wrong places.

How can scientists tell STR loci apart in multiplexing?

Scientists can tell loci apart by fragment size and by using different fluorescent dyes. This means even fragments that overlap in size can still be distinguished by colour.

What is fluorescent DNA analysis technology?

Fluorescent DNA analysis uses fluorescent tags attached to DNA fragments. A scanner detects the fluorescence as fragments pass through, allowing the fragments to be identified and measured.

What is an electropherogram?

An electropherogram is a graph showing DNA fragment peaks. Each peak represents a DNA fragment, and the position of the peak relates to fragment size.

What does peak position show on an electropherogram?

Peak position shows the size of the DNA fragment. From this, scientists can work out the STR repeat number.

What does peak height show on an electropherogram?

Peak height shows the amount of DNA signal. Taller peaks usually mean more DNA of that fragment was detected.

How do you get a genotype from an electropherogram?

You estimate the size of the PCR fragment, subtract the flanking region size, then divide the remaining repeat region by the repeat unit length. This gives the repeat number, which is the genotype at that locus.

What are flanking regions?

Flanking regions are the DNA sequences on either side of the STR repeat region. PCR primers bind to these regions so the STR can be copied.

Why do you subtract the flanking regions when working out repeat number?

The total PCR fragment includes both the STR repeat region and the flanking regions. To find the number of repeats, you need to remove the flanking region length first.

Why do STR genotypes usually produce one or two numbers?

One number means both alleles are the same, so the person is homozygous. Two numbers mean the person has two different alleles, so they are heterozygous.

What does allele calling mean?

Allele calling is the process of deciding which repeat number a DNA peak represents. It requires comparing the peak size to known standards and making a judgement.

Why can allele calling sometimes involve judgement?

Some peaks may be weak, unclear, or affected by artefacts. Scientists must decide whether a peak is a real allele or not, which is why interpretation standards are important.

What are off-ladder alleles?

Off-ladder alleles are alleles that do not match the standard allele ladder used for comparison. They may require expert judgement to interpret.

Why are STR marker panels used internationally?

Many countries use overlapping STR loci, which allows DNA profiles to be compared across different databases and jurisdictions.

Why is broad international comparability useful?

It allows DNA profiles generated in different places to be compared more easily, because many of the same STR loci are being tested.

What is the main advantage of STR analysis over older VNTR analysis?

STR analysis needs less DNA, is faster, works better with degraded samples, and can be automated more easily.

What is the power of PCR in forensic DNA analysis?

PCR allows very small or degraded samples to be analysed by copying specific DNA regions many times.

What is the main advantage of automation in STR analysis?

Automation allows many STR loci to be analysed more quickly and consistently. It also helps produce electropherograms that can be interpreted systematically.

Why is interpretation still important even with automated STR analysis?

The machine produces data, but scientists still need to interpret the peaks, check for artefacts, consider mixtures, use population data, and explain the statistical meaning of the result.

What is the overall legal importance of DNA evidence?

DNA is a very powerful forensic tool, but its strength depends on sample quality, lab standards, correct interpretation, and accurate statistical databases.