alternative markers

What are the two main forensic applications of capillary electrophoresis?

DNA fragment analysis and DNA sequence analysis.

What is capillary electrophoresis (CE)?

An electrophoresis technique that separates DNA fragments using a polymer sieving medium inside a capillary.

What medium does capillary electrophoresis use instead of a traditional gel?

A polymer sieving medium.

What are the advantages of capillary electrophoresis?

Reduced preparation time, increased sensitivity and the ability to process samples using 96- or 384-well plates.

Why does capillary electrophoresis require less preparation than gel electrophoresis?

There is no need to pour a gel or load samples into small gel wells.

What is the purpose of fluorescent detection in DNA analysis?

To detect fluorescently labelled DNA fragments or sequencing products.

What equipment is used in fluorescent detection?

A laser, camera, computer and specialist software.

What is the function of the laser in fluorescent detection?

It excites the fluorescent labels attached to DNA.

What is the function of the camera in fluorescent detection?

It captures the emitted fluorescent signal.

What is spectral calibration?

A process that allows software to distinguish fluorescent dyes by correcting for spectral overlap.

Why is spectral calibration necessary?

Different fluorescent dyes may have overlapping emission spectra, which can interfere with accurate detection.

Is spectral calibration the same for every dye set?

No, calibration is unique to each dye set.

What is Sanger sequencing?

A DNA sequencing method that determines the order of nucleotide bases using chain-terminating dideoxynucleotides.

Who developed Sanger sequencing and when?

Frederick Sanger and colleagues developed the method in 1974.

What is the alternative name for Sanger sequencing?

Dideoxy sequencing or chain-termination sequencing.

What are ddNTPs?

Dideoxynucleotides that lack the 3'-hydroxyl group needed for continued DNA chain extension.

What is the function of dNTPs in Sanger sequencing?

They allow DNA polymerase to extend the growing DNA strand.

What is the function of ddNTPs in Sanger sequencing?

They terminate DNA strand extension when incorporated.

Why do ddNTPs terminate DNA synthesis?

They lack the 3'-OH group needed to form the next phosphodiester bond.

What is required for a Sanger sequencing reaction?

Reaction buffer, DNA polymerase, sequencing primer, dNTPs, fluorescently labelled ddNTPs and DNA template.

Where does the Sanger sequencing reaction take place?

In a PCR machine.

Why is a second PCR stage required in the Sanger sequencing process described?

To carry out the sequencing reaction using the previously amplified DNA template.

Why are ddNTPs fluorescently labelled?

So the identity of the terminating nucleotide can be detected.

What happens when DNA polymerase adds a normal dNTP?

DNA chain extension continues.

What happens when DNA polymerase adds a ddNTP?

DNA chain extension terminates.

What does Sanger sequencing produce?

DNA fragments of different lengths, each ending with a fluorescently labelled ddNTP.

How are DNA fragments separated during sequencing?

By electrophoresis according to their size.

How do adjacent sequencing fragments differ in length?

They generally differ by one base pair.

How is the DNA sequence determined?

The fluorescent labels of separated fragments are detected and used to identify the order of nucleotide bases.

What is a single nucleotide polymorphism (SNP)?

A variation at a single nucleotide position in the DNA sequence.

How do SNPs arise?

Through a single-base change or point mutation in the genome.

Where are SNPs found?

Throughout the human genome.

Approximately how many SNPs have been detected in the human genome according to the lecture?

Approximately 38 million.

Approximately how many SNPs may an individual have according to the lecture?

Approximately 3.6 million.

What is a bi-allelic SNP?

A SNP with two possible nucleotide variants.

Can SNPs have more than two alleles?

Yes, tri-allelic and tetra-allelic SNPs have also been identified.

What are the main advantages of SNP amplification?

Very short target fragment lengths, no stutter products and the ability to multiplex.

Why are SNPs useful when analysing degraded DNA?

Their short target fragment lengths make them more suitable for amplifying fragmented DNA.

Do SNP profiles produce stutter products like STR profiles?

No, SNP analysis does not produce the typical STR stutter products.

What is multiplexing?

Analysing multiple genetic markers in a single reaction.

What is SNaPshot analysis?

A technique used to identify SNPs through a single-base extension reaction followed by capillary electrophoresis.

What are the main stages of SNaPshot-CE analysis?

Amplify around the target SNP, post-PCR clean-up, carry out a sequencing or single-base extension reaction, then analyse using capillary electrophoresis.

What are identity SNPs?

SNP markers used to distinguish between individuals.

What is the purpose of identity SNPs in forensic science?

To help identify or exclude individuals as possible sources of biological evidence.

What is heterozygosity?

The presence of two different alleles at a particular genetic locus.

Why is high heterozygosity desirable when selecting identity SNPs?

It maximises the amount of distinguishing information provided by each marker.

What is Fst?

A measure of genetic differentiation between populations.

Why is low Fst desirable when selecting identity SNPs?

It reduces the likelihood that marker differences are strongly influenced by population structure.

How many SNPs are approximately equivalent to 12 STRs in the lecture?

Approximately 50 SNPs.

Why are known DNA profiles useful when using identity SNPs?

The unknown profile needs a reference profile for comparison and identification.

What is forensic DNA phenotyping (FDP)?

The prediction of externally visible characteristics of an unknown individual using DNA or other molecular biomarkers.

What does EVC stand for?

Externally visible characteristic.

What is the purpose of forensic DNA phenotyping?

To provide investigative intelligence about the likely appearance of an unknown DNA donor.

What is the difference between DNA profiling and DNA phenotyping?

DNA profiling compares genetic profiles to identify or exclude individuals, whereas phenotyping predicts characteristics such as eye, hair and skin colour.

Which human pigment traits can be predicted using SNPs?

Eye colour, hair colour and skin colour.

Why are pigment traits easier to predict than facial features?

Pigment traits are generally less genetically complex than characteristics such as facial morphology.

What is the HIrisPlex system?

A DNA-based system used to predict hair and eye colour.

What is the HIrisPlex-S system?

A system that predicts eye, hair and skin colour from DNA.

How many SNP markers are used by HIrisPlex-S?

41 SNP markers.

How many eye colour categories can HIrisPlex-S predict according to the lecture?

Three categories.

How many hair colour categories can HIrisPlex-S predict according to the lecture?

Four categories.

How many skin colour categories can HIrisPlex-S predict according to the lecture?

Five categories.

What minimum DNA quantity was associated with obtaining a full HIrisPlex-S profile in the lecture?

63 pg.

What is a limitation of predicting skin colour from DNA?

Predictions provide probabilities rather than a guaranteed description of an individual's appearance.

Why are complex EVCs difficult to predict?

They involve many genes, complex inheritance patterns and environmental influences.

Why is height difficult to predict accurately from DNA?

Many genes contribute to height, environmental factors influence it and much genetic information remains unknown.

What genetic factors are associated with early-onset male pattern baldness?

Variants in several genes and genomic regions, particularly the AR/EDA2R region on the X chromosome.

What is DNA methylation?

A chemical modification of DNA that can influence gene activity without changing the underlying DNA sequence.

Why is DNA methylation relevant to age estimation?

Changes in DNA methylation patterns can be used to estimate an individual's biological age.

What is facial morphology?

The structure, shape and features of the human face.

Why is facial morphology difficult to predict from DNA?

Many genetic variants contribute small effects, and facial appearance is also influenced by sex and genetic ancestry.

What characteristics can Parabon Snapshot attempt to predict?

Sex, ancestry, skin pigmentation, hair colour, eye colour, freckling and facial morphology.

What are ancestry-informative SNPs?

SNP markers used to provide information about an individual's genetic ancestry.

Why are SNPs useful for ancestry analysis?

They have relatively low mutation rates and some variants occur at different frequencies in different populations.

What is the approximate SNP mutation rate stated in the lecture?

Approximately one mutation per 10^8 generations.

What is the approximate STR mutation rate stated in the lecture?

Approximately one mutation per 1,000 generations.

What does it mean when a SNP is fixed in a population?

A particular allele is present at a very high frequency or is effectively the only observed allele in that population.

Can ancestry SNPs establish an individual's exact ethnicity?

No, they provide probabilistic information about genetic ancestry and cannot establish an exact identity or ethnicity.

What is the Y chromosome?

A sex chromosome that is generally passed from father to son.

How is the Y chromosome inherited?

Paternally, from father to son.

What is the approximate size of the Y chromosome discussed in the lecture?

Approximately 60 megabases.

What is the SRY gene?

The sex-determining region Y gene, which plays a key role in testes development.

What are Y-STRs?

Short tandem repeat markers located on the Y chromosome.

What are Y-SNPs?

Single nucleotide polymorphisms located on the Y chromosome.

What is Y-Filer Plus?

A commercial Y-STR amplification kit used for forensic analysis.

How many Y-STR loci does Y-Filer Plus analyse?

27 Y-STR loci.

How many rapidly mutating Y-STRs are included in Y-Filer Plus?

Seven.

What is a rapidly mutating Y-STR?

A Y-STR marker with a relatively high mutation rate, allowing greater differentiation between some related males.

What mutation rate is associated with the rapidly mutating markers discussed in the lecture?

Above 1%.

What is one advantage of Y-STR analysis in sexual assault investigations?

It specifically targets male DNA, which can help detect a male contributor in a large excess of female DNA.

What is another advantage of Y-STR analysis?

It uses a similar analysis process to autosomal STR profiling.

How can rapidly mutating Y-STRs improve identification?

They can improve discrimination between some related male individuals.

What is a major limitation of Y-STR profiling?

Closely related males may share the same or very similar Y-STR haplotype.

Why can Y-STR profiles have limited discriminatory power between relatives?

The Y chromosome is inherited along the paternal line, so male relatives may share the same haplotype.

Can Y-STR profiles be directly searched against the National DNA Database in the same way as standard autosomal STR profiles?

No, they cannot be directly compared with the standard NDNAD profiles.

Why is a known suspect useful for Y-STR comparison?

A reference Y-STR profile is needed for direct comparison with the evidential profile.

What is the Y-Chromosome Haplotype Reference Database (YHRD)?

A database containing Y-chromosome haplotype information used to estimate population frequencies.

What is a Y-STR haplotype?

A combination of Y-STR alleles inherited together along the paternal lineage.

What is the purpose of a Y-chromosome elimination database?

To collect Y-chromosome profiles from relevant individuals, such as personnel, to help identify or eliminate possible sources of DNA.

What ethical concerns may arise from a Y-chromosome elimination database?

Privacy, informed consent, data security, familial implications, retention of genetic information and potential misuse.