Forensic Biology Processes and Tests

types of blood tests

• phenolphthalein

• hemastix

• hemochromogen

explain a presumptive test

• screening test used to detect the possibility of a certain biological fluid

• can be positive or negative but positive doesn’t confirm the identity

• cheap and quick way to narrow down stains prior to other types of analyses

explain a confirmatory test

• follow up test to presumptive that is used to verify the identity of a biological fluid

• more specific & accurate than presumptive

• a positive result confirms the presence of a substance

phenolphthalein assay

• presumptive test for blood

• phenolphthalein is a colorless compound catalyzed by heme using hydrogen peroxide as the oxidant

• once oxidized it turns pink in alkaline conditions

• strong oxidants can cause false positives (bleach, hair dye)

• plant peroxidases can catalyze rxn and cause false positives (horseradish)

hemochromogen crystal test

• confirmatory test for blood

• stain treated w/pyridine and glucose to form crystals of pyridine ferroprotoporphyrin

• blood specific not human specific

hemastix test

• presumptive test for blood

• positive result is a chemical indication of blood

• detects peroxidase like activity of hemoglobin through color change

• not human specific

Acid phosphatase

• presumptive test for seminal fluid

• catalyzes removal or phosphate group from the substrate, insoluble colored precipitate forms w/diazonium salts

• if AP is present alpha-napthyl phosphate is hydrolyzed to phosphate and alpha-napthol

• alpha-napthol combines w/fast blue B to produce purple color

• false positives can include fruit and vegetable juices, oral, vaginal secretions

ABAcard p30

• confirmatory test for semen, immunochromatographic test

• detects p30 in semen

• sensitive as low as 4ng/ml

• subject to high dose hook effect

• antibody specifically binds to p30 antigen in seminal fluid

christmas tree stain

• confirmatory test for semen

• nuclear fast red stain nuclei of spermatozoa, nuclei & acrosomal cap pink/red

• picroindigocarmine stains the neck and tail green

• epithelial cells will be blue green

• not human specific

extraction

process of breaking open (lysing) cells to release DNA from biological material

low molecular weight dna

• dna fragments that are smaller and have a low number of base pairs

• environmentally exposed samples

• hair shafts

• ancient dna

• touch dna

high molecular weight dna

• blood

• tissue

• semen

• saliva

• bone marrow

• reference swabs

inhibition

prevention of DNA amplification by binding to ss or ds DNA. Interfere w/cell lysis

common inhibitors

• hemoglobin

• melanin

inhibitors negatively affect quant and amp

sodium dodecyl sulfate (SDS)

• commonly used in extractions

• detergent w/hydrophilic head & hydrophobic tail

• SDS inserts into phospholipid bilayer and breaks membrane

proteinase K (proK)

• enzyme that digests proteins

• function stimulated by SDS

• specifically needed to digest proteins that form complex w/DNA = histones

• need to free DNA from cell and from proteins

EDTA

• chelating agent

  • binds to divalent metal ions

• protect DNA from degradation during extraction

• can help disrupt the cell membrane and assist in extracting DNA

DTT

• dithiothreitol

• breaks protein disulfide bridges present in some sample types

• disulfide bridges have strong bonds that cannot be broken with SDS & proK

• DTT must be added during lysis step of extraction for these samples

quantitation

• determining the concentration of DNA in sample

DNA isolation

• organic solvents added to sample mixed and centrifuged

• centrifuge allows layers to form

• aqueous layer containing DNA is removed and washed w/solvents to isolate DNA

dna purification

• large volume of aqueous phase = low conc of DNA

• centrifugal filter allows for purification and concentration

• DNA sticks to filter while other components are pulled through

Reagent blank

• type of control

• contains all of the chemicals added during extraction

• used to ensure no contamination during the extraction process

• if control fails, samples are invalid

why do we quant dna

1. FBI quality assurance standards

2. do we have human dna

3. troubleshooting

4. pcr kits are optimized in certain ranges of concentration 0.5 - 2 ngs

real time pcr

• accurately determine quality and quantity of DNA in a sample

• works by measuring cycle to cycle changes in florescent signal from amplification of the target

• compares those fluorescence signals from unknown samples to the signals of standards

polymerase chain reaction

• replicates specific region of DNA during repetitive cycles making many copies for downstream processing

• heating and cooling cycling pattern

• target defined by primers that are complementary to sequence of interest

taqman probe chem

• probe annealed to template DNA contains two fluorescent dyes that emit fluorescence at different wavelengths

• when dyes are close to each other fluorescence suppressed

• during polymerase activity probe will become displaced and dye will fluroesce

• amount of fluorescence is proportional to DNA in the sample

IPC

• internal positive control

• makes sure instrument and chemistry is working as expected

• undergoes same process as template DNA

dNTPs

• deoxyribonucleotide triphosphates

• building blocks

• high concentrations may promote misincorporations

master mix

used

• homogeneity

• reproducibility

• reduces sample to sample

• improves accuracy

pcr controls

• positive and negative controls amped concurrently w/associated samples

  • same kit

  • same instrument

  • same time

• positive control: synthetic DNA with known profile

  • shows amplification reagents and equipment is working properly

extraction blank

• assesses contamination introduced during the entire sample preparation process, including extraction

what is a dna profile

• unique set of genetic markers used to identify individuals

• analyze specific variable regions of an individuals DNA

allelic ladder

• sample of DNA fragments which contain all alleles at each loci

• DNA fragments of known sizes

• use the same primers as the test samples

• used for accurate allele determination

ILS internal lane standard

• DNA fragments of known sizes that is run with the sample DNA to serve as a size reference

• helps to convert the time it took to come off the capillary to base pairs

chain of custody

• detailed log of the history of the evidence including collection, storage, and analysis

• ensures evidence integrity

why do we amp DNA

• for detection and downstream analysis

• easier to visualize and study specific DNA segments

why does the nist database apply to my client

• NIST database is required and validated by my labs SOPs

• why they only chose those populations I cannot speak for NIST

what is the random match probability

the probability that if you picked a random unrelated person from the population and their profile matching the profile recovered from the evidence

explain dna extraction

• the sample is added to a special tube chemicals break open the cells and release the DNA

• another chemical is added to help the dna stick to the membrane in the tube

• the tube is spun so the liquid is separated from the DNA and debris is washed away

explain dna quantitation

• measuring how much DNA in a sample

• used pcr to measure the amount of DNA by using signals

• compare unknown signals to standard signals

explain dna amplification

• make a solution to target 0.5 ng of DNA

• place sample in thermocycler

• making more copies of a specific region of DNA

what is capillary electrophoresis

• process that separates dna fragments based on size

• fragments pass a detector and it records their size and color

• used to develop dna profiles