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ABC Forensic DNA practice questions

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Question 1Biological Evidence Screening and Sample Selection

Which statement best describes the luminol test used during biological evidence screening?

  1. Detects saliva by amylase activity
  2. Detects blood by chemiluminescence Correct answer.
  3. Detects urine by creatinine
  4. Detects semen by acid phosphatase

Explanation

Luminol produces light when it reacts with the iron in hemoglobin, making it useful for locating latent bloodstains. It is a presumptive test, so a positive result must be followed by confirmatory testing and DNA analysis where appropriate.

Question 2Biological Evidence Screening and Sample Selection

Which test is considered confirmatory for the presence of blood?

  1. Leucomalachite test
  2. Kastle-Meyer test
  3. Takayama test Correct answer.
  4. Luminol test

Explanation

The Takayama test forms characteristic heme crystals and is used as a confirmatory test for blood. Kastle-Meyer, luminol, and leucomalachite green are presumptive tests that indicate the possible presence of blood.

Question 3Biological Evidence Screening and Sample Selection

Which reagent is commonly used in the acid phosphatase test for suspected semen?

  1. Amido black stain
  2. Leucomalachite green
  3. Brentamine Fast Blue Correct answer.
  4. Luminol spray

Explanation

Acid phosphatase hydrolyzes phosphate esters, and the reaction with a diazonium dye such as Brentamine Fast Blue produces a colored product. This is a presumptive test for semen and should be followed by confirmatory or DNA analysis.

Question 4Biological Evidence Screening and Sample Selection

The Christmas tree stain is used to identify which biological material?

  1. Urine stains
  2. Bloodstains
  3. Spermatozoa Correct answer.
  4. Saliva stains

Explanation

The Christmas tree stain colors sperm heads red and tails green, allowing microscopic identification of spermatozoa. It is commonly used to confirm the presence of sperm in sexual assault evidence.

Question 5Biological Evidence Screening and Sample Selection

What is the recommended handling of a swab containing a dried biological stain before packaging?

  1. Seal wet in plastic
  2. Freeze while wet
  3. Soak in bleach
  4. Air-dry before packaging Correct answer.

Explanation

Swabs should be air-dried at room temperature before packaging to reduce moisture and microbial growth that can degrade DNA. Wet packaging can promote bacterial proliferation and sample deterioration.

Question 6Biological Evidence Screening and Sample Selection

Which sample is most commonly collected as a DNA reference from a living person?

  1. Hair shaft
  2. Buccal swab Correct answer.
  3. Fingernail clipping
  4. Urine sample

Explanation

A buccal swab collects epithelial cells from the inside of the cheek and provides a reliable source of reference DNA. Hair shaft without root, nail clippings, and urine are not preferred reference sources.

Question 7Biological Evidence Screening and Sample Selection

What is the primary purpose of an alternate light source during evidence screening?

  1. Measuring DNA quantity
  2. Assessing DNA degradation
  3. Determining DNA sequence
  4. Finding biological stains Correct answer.

Explanation

Alternate light sources can cause certain biological fluids and materials to fluoresce or absorb light differently, helping examiners locate stains. They do not measure DNA quantity, sequence DNA, or assess degradation.

Question 8Biological Evidence Screening and Sample Selection

The Phadebas test detects which enzyme to indicate the possible presence of saliva?

  1. Urease
  2. Alpha-amylase Correct answer.
  3. Catalase
  4. Acid phosphatase

Explanation

Phadebas is a presumptive test for alpha-amylase, an enzyme found in saliva. A positive result supports further testing for saliva but does not by itself prove the fluid's origin.

Question 9Biological Evidence Screening and Sample Selection

When sampling a cigarette butt for saliva DNA, which area should be targeted?

  1. Paper wrapper
  2. Outer cellophane wrapper
  3. Ash-covered end
  4. Filter end Correct answer.

Explanation

The filter end is the area that contacts the mouth and is most likely to contain saliva-derived cells. Sampling the ash end or outer wrappers is less likely to recover informative DNA.

Question 10Biological Evidence Screening and Sample Selection

A dark fabric is suspected to contain a semen stain that is not visible under normal light. Which screening approach is most appropriate?

  1. Amylase test
  2. Luminol spray
  3. Acid phosphatase
  4. ALS examination Correct answer.

Explanation

Alternate light sources can reveal fluorescence or contrast differences on dark or patterned fabrics where stains are not visible. Luminol is for blood, while acid phosphatase and amylase are chemical screening tests that may follow visual or ALS localization.

Question 11Biological Evidence Screening and Sample Selection

What is the purpose of differential extraction in sexual assault evidence?

  1. Amplify STR markers
  2. Increase total DNA yield
  3. Remove inhibitory substances from extract
  4. Separate sperm and epithelial cells Correct answer.

Explanation

Differential extraction separates sperm cells from epithelial cells so that each fraction can be analyzed separately. This helps resolve mixtures and improves interpretation of sperm and non-sperm DNA contributions.

Question 12Biological Evidence Screening and Sample Selection

A swab from a sexual assault kit is sealed in a closed tube while still wet. What is the most likely consequence?

  1. Increases sperm motility
  2. Prevents any DNA degradation
  3. Preserves RNA intact
  4. Promotes bacterial growth Correct answer.

Explanation

Moisture in a sealed container creates an environment that supports microbial growth and can accelerate DNA degradation. Swabs should be air-dried before packaging whenever possible.

Question 13Biological Evidence Screening and Sample Selection

A semen stain is present on a piece of clothing. Which sampling method is generally preferred for DNA analysis?

  1. Tape lift the fabric
  2. Scrape with a blade
  3. Cut stained area Correct answer.
  4. Swab the dry surface

Explanation

Cutting out the stained area preserves the biological material and allows the laboratory to sample the stain directly. Swabbing, scraping, or tape lifting may be useful for trace or surface samples but can be less effective for a visible fabric stain.

Question 14Biological Evidence Screening and Sample Selection

A presumptive blood test is positive on a rusty metal surface. What is the most appropriate next step?

  1. Confirm and sample the stain Correct answer.
  2. Assume it is human blood
  3. Treat it as a semen stain
  4. Assume it is human blood without testing

Explanation

Rust and other oxidants can cause false-positive presumptive blood reactions, so a positive result should be followed by confirmatory testing and appropriate sampling. The sample can then be analyzed for DNA if sufficient material is present.

Question 15Biological Evidence Screening and Sample Selection

What does the PSA test detect in forensic screening?

  1. Urine
  2. Saliva
  3. Blood
  4. Semen Correct answer.

Explanation

Prostate-specific antigen is a semen marker used in presumptive screening for seminal fluid. It is not a blood, saliva, or urine marker.

Question 16Biological Evidence Screening and Sample Selection

Which hair sample is most suitable for nuclear DNA analysis?

  1. Hair shaft
  2. Distal tip
  3. Mid-shaft segment
  4. Root with sheath Correct answer.

Explanation

The root with attached sheath contains follicular cells and is the best hair portion for nuclear DNA analysis. The hair shaft and distal tip generally contain limited nuclear DNA.

Question 17Biological Evidence Screening and Sample Selection

Which skeletal sample is generally preferred for DNA analysis?

  1. Cartilage tissue
  2. Hair shaft
  3. Dense cortical bone Correct answer.
  4. Spongy cancellous bone

Explanation

Dense cortical bone is relatively resistant to environmental damage and often yields better-preserved DNA than spongy bone. Cartilage and hair shaft are not preferred skeletal DNA sources.

Question 18Biological Evidence Screening and Sample Selection

Tape lifting is most appropriate for collecting which type of evidence?

  1. Fresh semen
  2. Liquid blood
  3. Touch DNA Correct answer.
  4. Liquid saliva

Explanation

Tape lifting can recover trace cellular material from surfaces where swabbing might be less effective. It is commonly used for touch DNA on fabrics or irregular surfaces, not for bulk liquids.

Question 19Biological Evidence Screening and Sample Selection

How should a buccal reference swab be collected?

  1. Rub inner cheek Correct answer.
  2. Touch the tongue
  3. Swab the gums
  4. Collect saliva in tube

Explanation

A buccal swab is collected by rubbing the inside of the cheek to recover epithelial cells. This provides a reliable reference sample with minimal invasiveness.

Question 20Biological Evidence Screening and Sample Selection

Which information is essential on the label of a collected swab?

  1. Item, date, initials Correct answer.
  2. Only the collector name
  3. Only the collection date
  4. Location and item type

Explanation

Evidence labels should uniquely identify the item, collection date, and collector or initials to maintain chain of custody. Complete labeling supports traceability from collection through analysis.

Question 21Biological Evidence Screening and Sample Selection

What is a key advantage of mRNA or miRNA markers for body fluid identification?

  1. Fluid-specific expression Correct answer.
  2. Higher total nuclear DNA yield
  3. Better STR peak heights
  4. Faster extraction time

Explanation

mRNA and miRNA markers can show fluid-specific expression patterns, allowing examiners to infer the type of body fluid present. They complement DNA typing rather than primarily increasing DNA yield or STR signal.

Question 22Biological Evidence Screening and Sample Selection

A stain gives a positive presumptive blood test but a negative Takayama test, and a DNA profile is still obtained. What is the best interpretation?

  1. The DNA result is invalid
  2. The stain is certainly blood
  3. The stain must be semen
  4. False positive is possible Correct answer.

Explanation

Presumptive tests can react with non-blood substances, so a negative confirmatory test suggests a possible false positive. The DNA profile remains valid because DNA analysis does not depend on the confirmatory blood result.

Question 23Biological Evidence Screening and Sample Selection

What is the best sampling strategy for low-template touch DNA on a large surface?

  1. Several targeted swabs Correct answer.
  2. Douse with water
  3. Scrape with a blade
  4. One swab of entire surface

Explanation

Low-template touch DNA is best recovered by targeting likely contact areas with multiple swabs rather than sampling the entire surface indiscriminately. Excess moisture and scraping can reduce recovery or damage the substrate.

Question 24Biological Evidence Screening and Sample Selection

After differential extraction of a mixed stain, which fraction is expected to contain sperm DNA?

  1. Sperm pellet fraction Correct answer.
  2. Initial wash buffer
  3. First lysate supernatant
  4. Epithelial supernatant

Explanation

In differential extraction, epithelial cells are lysed first and separated, while sperm cells remain in the pellet and are lysed subsequently. The sperm pellet fraction is therefore enriched for sperm DNA.

Question 25Biological Evidence Screening and Sample Selection

Which practice best reduces contamination during evidence screening?

  1. Use one scissors throughout
  2. Talk over open samples
  3. Reuse the same swab
  4. Change gloves between items Correct answer.

Explanation

Changing gloves between items reduces the transfer of DNA from one sample to another. Reusing tools, talking over open samples, and using the same scissors without cleaning increase contamination risk.

Question 26Biological Evidence Screening and Sample Selection

What is the main purpose of a presumptive test during biological evidence screening?

  1. To give a legally conclusive identification of a fluid
  2. To name the donor before extraction is attempted
  3. To flag areas that may contain body fluid for sampling Correct answer.
  4. To measure how much DNA is present in a stain

Explanation

Presumptive tests are rapid screening tools; a positive result only suggests that a particular body fluid may be present and helps the analyst decide which areas to sample. Confirmation requires a separate, more specific test, and donor identity is established later by DNA profiling.

Question 27Biological Evidence Screening and Sample Selection

A luminol spray produces a brief blue glow on a wall that appears clean. What does this result most likely indicate?

  1. Seminal fluid is definitely present on the wall
  2. DNA from the person who cleaned the wall is present
  3. Only household bleach is present on the wall
  4. Blood may be present; the area should be sampled Correct answer.

Explanation

Luminol emits light when it is oxidized in the presence of hemoglobin, so a glow indicates that blood may be present even when the stain cannot be seen. The reaction is presumptive and can also be produced by bleach, metal ions, and some plant materials, so the area must be sampled and confirmed by other tests.

Question 28Biological Evidence Screening and Sample Selection

Which reagent is used in the common presumptive test for seminal fluid based on acid phosphatase activity?

  1. Leucomalachite green
  2. Brentamine Fast Blue B Correct answer.
  3. Luminol working solution
  4. Hexagon OBTI strip

Explanation

Seminal fluid is rich in acid phosphatase, and Brentamine Fast Blue B reacts with the enzyme product to form a colored azo dye. The test is presumptive because acid phosphatase also occurs in other body fluids and in some plant materials.

Question 29Biological Evidence Screening and Sample Selection

Which presumptive blood test depends on the peroxidase-like activity of hemoglobin to oxidize a colorless dye?

  1. Kastle–Meyer test Correct answer.
  2. Brentamine test
  3. Teichmann test
  4. Takayama test

Explanation

In the Kastle–Meyer test, hemoglobin catalyzes the oxidation of reduced phenolphthalein by hydrogen peroxide, producing a pink color. The Teichmann and Takayama tests instead form hemin crystals and serve as confirmatory tests.

Question 30Biological Evidence Screening and Sample Selection

A blood-like stain is found on a varnished wooden floor. In addition to the stain, what should be collected?

  1. A swab of the analyst's glove, to monitor laboratory contamination
  2. A second sample from the stain center, to increase DNA yield
  3. A sample of the cleaning agent, to test for bleach
  4. An unstained piece of the same wood, as a control Correct answer.

Explanation

Substrate controls show whether the material itself reacts with the screening reagent or interferes with later testing. Varnished and chemically treated woods can produce false-positive peroxidase reactions, so an unstained area of the same wood is tested alongside the stain.

Question 31Biological Evidence Screening and Sample Selection

Which collection technique is recommended for recovering dried epithelial DNA from human skin, such as around a bite mark?

  1. Repeated tape lifts pressed onto the skin surface
  2. One dry swab rubbed firmly over the area for a minute
  3. A moistened swab followed by a dry swab over the same area Correct answer.
  4. An adhesive film left on the skin overnight

Explanation

The double-swab technique uses a swab moistened with sterile water to rehydrate and lift dried cells, followed by a dry swab to recover the remaining moisture and cellular material. Both swabs are then air-dried and submitted together.

Question 32Biological Evidence Screening and Sample Selection

A complainant reports only oral contact. Which kit samples are most probative for DNA?

  1. Vaginal swabs collected at the same examination
  2. Oral swabs collected within the recommended timeframe Correct answer.
  3. Fingernail clippings from both hands
  4. Rectal swabs collected at the same examination

Explanation

Sample selection should follow the reported acts and the likely deposition sites, so oral contact is best addressed with oral swabs taken as soon as practicable. Samples from sites not involved in the account add little probative value and can complicate mixture interpretation.

Question 33Biological Evidence Screening and Sample Selection

A dried bloodstain is on a large, immovable metal door frame. Which collection approach is generally preferred?

  1. Scrape the dried stain into a paper envelope
  2. Lift the stain with adhesive tape onto a slide
  3. Swab the stain with a water-moistened sterile swab Correct answer.
  4. Cut a section of the metal frame and submit it

Explanation

When an item cannot be transported, a moistened swab is used to rehydrate and transfer the dried stain, and the swab is air-dried before packaging. Scraping and tape lifting tend to recover less DNA and can introduce contaminants or lose material.

Question 34Biological Evidence Screening and Sample Selection

Before sampling a stain on a garment, which step is most important for the case record?

  1. Wash the garment thoroughly to remove any dyes
  2. Outline the stain with a permanent marker
  3. Photograph the stain with a scale and label it Correct answer.
  4. Estimate the DNA yield from the stain's size

Explanation

Documentation preserves the location, appearance, and size of the stain before sampling alters or removes it, which supports later interpretation in court. Photographs should include a scale and an item identifier.

Question 35Biological Evidence Screening and Sample Selection

Why is a known reference sample collected from a complainant in a sexual assault case?

  1. To replace the need for DNA extraction from the stains
  2. To measure the amount of semen in the kit swabs
  3. To confirm that the complainant was present at the scene
  4. To distinguish the complainant's DNA from foreign DNA Correct answer.

Explanation

A known profile from the complainant allows foreign alleles in a mixture to be identified and subtracted during interpretation. Reference samples are compared with, not substituted for, the evidentiary samples.

Question 36Biological Evidence Screening and Sample Selection

After applying a buccal sample to an FTA card, what should be done before packaging?

  1. Seal the card in a plastic bag while still damp
  2. Allow the card to air-dry completely Correct answer.
  3. Store the card at −80 °C immediately
  4. Rinse the card with ethanol to remove inhibitors

Explanation

FTA cards must be thoroughly air-dried so that the treated paper can lyse cells and preserve DNA without moisture-driven degradation or mould. Once dry, the card can be packaged and stored at room temperature.

Question 37Biological Evidence Screening and Sample Selection

A stain gives a strong presumptive blood reaction, but DNA extraction yields no profile. Which explanation is most consistent?

  1. The extraction batch failed for every sample in the run
  2. Blood residues may persist after DNA has degraded Correct answer.
  3. Blood is not a source of amplifiable human DNA
  4. The presumptive test must have been a false positive

Explanation

Hemoglobin and its breakdown products can remain detectable long after the DNA in a stain has been degraded by heat, moisture, sunlight, or microbial action. A positive screening result therefore does not guarantee a profile, and other samples in the same batch may have amplified normally.

Question 38Biological Evidence Screening and Sample Selection

A vaginal swab gives a positive PSA result but no spermatozoa are seen under the microscope. Which explanation is most likely?

  1. Spermatozoa are only visible after hematoxylin staining
  2. The swab was collected from the wrong anatomical site
  3. The donor may be azoospermic or have had a vasectomy Correct answer.
  4. PSA is produced by vaginal epithelial cells

Explanation

PSA is a seminal fluid protein, so a positive result indicates seminal fluid even when no sperm are present. Azoospermia, vasectomy, or degradation of sperm can explain the negative microscopy, and DNA testing should still be attempted.

Question 39Biological Evidence Screening and Sample Selection

A swab was already used to perform an acid phosphatase test on a stain. Which approach is most appropriate if DNA testing is still needed?

  1. Extract it with additional washes, expecting possible inhibition Correct answer.
  2. Retest it with a confirmatory reagent before extraction
  3. Use it directly; the reagent does not affect DNA
  4. Discard it, because tested swabs cannot yield DNA

Explanation

Reagents used in presumptive tests can carry over and inhibit amplification, so a tested swab is a compromised but often still workable sample. Additional washing or a dedicated extraction protocol can reduce inhibitor effects, and a separate untreated sample is always preferable when one is available.

Question 40Biological Evidence Screening and Sample Selection

A stain screens negative with a presumptive test but is still submitted for DNA analysis. Which statement best justifies this decision?

  1. Extraction removes the need for any body fluid identification
  2. Screening can miss low-level material that still yields DNA Correct answer.
  3. Negative screening results are always false and can be ignored
  4. DNA analysis identifies body fluids that chemical tests cannot

Explanation

Presumptive tests have detection limits and can be blocked by degradation, dilution, or inhibitors, so a negative result does not prove that DNA is absent. Sampling decisions should weigh the case circumstances and the realistic chance of recovering a profile.

Question 41DNA Extraction, Quantitation, and Amplification

During enzymatic lysis in a forensic DNA extraction, what is the main function of Proteinase K?

  1. It removes RNA by hydrolyzing phosphodiester bonds in ribonucleotides.
  2. It chelates divalent cations such as Mg2+ that would activate DNases.
  3. It cleaves DNA at specific restriction sites to fragment the genome.
  4. It digests proteins and inactivates nucleases. Correct answer.

Explanation

Proteinase K is a broad-specificity serine protease that degrades proteins, including histones, freeing DNA from chromatin and destroying nucleases that would otherwise cut the template. It has no activity against nucleic acids, so it neither removes RNA nor fragments DNA. Removal of divalent cations is accomplished by chelating agents such as EDTA, not by the protease.

Question 42DNA Extraction, Quantitation, and Amplification

Which statement best describes how Chelex-100 protects DNA during a rapid boiling extraction?

  1. It binds DNA selectively while proteins flow through the column.
  2. It chelates cations that would otherwise degrade DNA. Correct answer.
  3. It acts as a chaotropic salt that lyses cells and denatures proteins.
  4. It is a fluorescent dye that intercalates between DNA base pairs.

Explanation

Chelex-100 is an ion-exchange resin that sequesters polyvalent metal ions such as Mg2+, which are required cofactors for DNases. With those cofactors removed, DNA survives the boiling step used to lyse cells, and the resin is then pelleted so the supernatant can be recovered. The resin does not bind DNA specifically and is not a chaotrope or a dye.

Question 43DNA Extraction, Quantitation, and Amplification

Human-specific forensic quantitation assays estimate the amount of amplifiable human DNA by targeting which kind of sequence?

  1. A human multicopy repeat element Correct answer.
  2. Plant chloroplast genes
  3. Bacterial 16S ribosomal RNA genes
  4. Yeast mitochondrial genes

Explanation

Human-specific quantitation targets multicopy human repeat elements, which occur in thousands of copies per genome and therefore give a strong, human-selective signal even from small samples. Prokaryotic, plant, and fungal sequences would report non-human DNA and are not used for this purpose. Targeting repeats also improves sensitivity compared with single-copy loci.

Question 44DNA Extraction, Quantitation, and Amplification

In the polymerase chain reaction, what is the role of the synthetic oligonucleotide primers?

  1. They catalyze phosphodiester bond formation between nucleotides.
  2. They supply the mononucleotide building blocks for the new strand.
  3. They separate the two strands of the template duplex.
  4. They define the ends of the product by binding flanking sequences. Correct answer.

Explanation

Primers are short single-stranded oligonucleotides that hybridize to sequences flanking the target, giving the polymerase a free 3'-hydroxyl from which to extend. They therefore set both the start and the end of the amplified product and confer specificity. Building blocks are provided by dNTPs, catalysis by the polymerase, and strand separation by heat.

Question 45DNA Extraction, Quantitation, and Amplification

In ethanol precipitation of DNA, why is a salt such as sodium acetate added before the ethanol?

  1. It increases the solubility of DNA in ethanol.
  2. It degrades residual protein remaining in the sample.
  3. It serves as a template for DNA synthesis.
  4. It shields backbone charges so DNA aggregates. Correct answer.

Explanation

The DNA backbone is polyanionic, and cations from the salt neutralize those negative charges so the molecules can come together and precipitate. DNA is insoluble in the resulting 60-70% ethanol solution, which allows it to be pelleted by centrifugation. Salt does not digest protein, enhance DNA solubility in ethanol, or act as a template.

Question 46DNA Extraction, Quantitation, and Amplification

In real-time PCR quantitation, what does the threshold cycle (Ct) value represent?

  1. The melting temperature of the amplified product.
  2. The total number of amplicons present at the endpoint.
  3. The cycle at which fluorescence first exceeds a set threshold. Correct answer.
  4. The number of cycles at which the reaction reaches its plateau.

Explanation

The Ct is the cycle number at which the fluorescence signal rises above a threshold set in the exponential phase, above background noise. Because the reaction is still exponential at that point, Ct is inversely proportional to the logarithm of the starting template amount. The plateau, melting temperature, and endpoint yield are separate measurements.

Question 47DNA Extraction, Quantitation, and Amplification

What is the purpose of an extraction negative control (reagent blank) in a forensic DNA laboratory?

  1. To measure the size of the amplified products
  2. To verify that the thermal cycler heats and cools correctly
  3. To confirm that the sample contains sufficient male DNA
  4. To detect DNA contamination from reagents or the process Correct answer.

Explanation

A reagent blank is carried through the same extraction and amplification steps as the evidence samples but contains no added sample. Any DNA profile obtained from it indicates contamination introduced by reagents, consumables, or handling, prompting investigation before results are reported. It is not a check on instrument temperature, amplicon sizing, or male DNA content.

Question 48DNA Extraction, Quantitation, and Amplification

A PCR is run with an annealing temperature well above the optimum calculated for its primers. What is the most likely result?

  1. Reduced primer binding and lower product yield Correct answer.
  2. More nonspecific amplification products
  3. Increased polymerase processivity
  4. Complete loss of primer dimers with unchanged yield

Explanation

Annealing temperature controls how stringently primers hybridize to their targets. Raising it above the optimum reduces stable primer-template duplex formation, so fewer primers are extended and product yield falls, sometimes to the point of complete failure. It is lowering the temperature that increases nonspecific products and primer dimers.

Question 49DNA Extraction, Quantitation, and Amplification

In differential extraction of a sexual assault sample, what does the dithiothreitol-containing lysis step accomplish?

  1. It removes male DNA while keeping the female fraction.
  2. It precipitates proteins from the sperm fraction.
  3. It selectively digests female epithelial DNA.
  4. It reduces disulfide bonds in sperm protamines. Correct answer.

Explanation

Sperm heads are stabilized by disulfide cross-links between protamine molecules, which make them resistant to ordinary lysis. Dithiothreitol reduces these bonds so the sperm heads can be opened and male DNA released into the sperm fraction. The earlier epithelial lysis step, without DTT, already released the female DNA.

Question 50DNA Extraction, Quantitation, and Amplification

Why is a fluorescent dye-binding assay often preferred over ultraviolet absorbance for quantifying forensic DNA extracts?

  1. It reports fragment length along with concentration.
  2. It measures concentration without any standards or calibration.
  3. It requires no sample and is non-destructive.
  4. The dye is DNA-selective, so RNA does not inflate results. Correct answer.

Explanation

UV absorbance at 260 nm responds to any nucleic acid as well as to some contaminants, so it can overestimate the amount of amplifiable DNA. Dye-based assays use fluorophores that fluoresce only when bound to double-stranded DNA, so RNA, free nucleotides, and most proteins do not contribute to the signal. These assays still require standards and a sample aliquot, and they do not report fragment length.