Forensic Biology Final
STRs normally consist of repeating sequences of:
3-7 bases.
Sexual Assualt Kit Components:
Biological Fluid Examinations Vaginal Specimen Anal Specimen Oral Specimen Fingernail Scrapings Dried Secretions/Debris Collection Trace evidence Pubic hair standards and combings Head hair standards and combings
Extreneous Peaks: Split peaks (+A/-A peaks)
Non-template addition results in a PCR product that is one base pair longer than the actual target sequence. When the polymerase is unable to complete the adenine addition on all products, this results in what is commonly referred to as split peaks (+A/-A peaks). To minimize split peaks, the extension phase of the PCR process is designed to drive the addition of adenine, ensuring that all amplicons are the same length.
______ is a very large molecule made by linking a series of repeating units called nucleotides.
Nuclear DNA strand
PCR Advantages/Disadvantages:
One advantage in moving to shorter DNA strands is that they would be expected to be more stable and less subject to degradation brought about by adverse environmental conditions. The long RFLP strands tend to readily break apart under the adverse conditions not uncommon at crime scenes. PCR also offers the advantage in that it can amplify minute quantities of DNA, thus overcoming the limited sample size problem often associated with crime scene evidence. PCR is best used with DNA strands that are no longer than a couple of hundred bases. With each amplification the DNA has the potential to be less perfect than the one before it. (It has the potential to be less perfect than the original.)
Single Source Profiles:
Profiles developed from a single contributor will display one or two alleles at each locus (excluding rare mutations). A heterozygous locus will show two peaks with roughly equal allele heights. A homozygous locus will show a single peak that is approximately twice the height of alleles seen at a heterozygous locus within the same dye color.
Components of Blood:
Red Blood Cells - Erythrocytes White Blood Cells - Leukocytes (WBCs have a nucleus. Useful for DNA analysis) Platelets - Thrombocytes Plasma - Clear, yellow tinted water (92%), sugar, fat, protein and salt solution which carries the red cells, white cells and platelets. 55% of blood's volume is made up of plasma. Antigens - usually proteins, located on the surface of red blood cells and are responsible for blood-type characteristics.
______ transport oxygen from the lungs to the body tissues and carry carbon dioxide back to the lungs.
Red blood cells
The position a gene occupies on a chromosome in known as a _______.
locus
The concept of simultaneously extracting, amplifying, and detecting a combination of STRs is known as:
multiplexing.
Extreneous Peaks: Spikes
narrow peaks usually attributed to fluctuation in voltage or the presence of minute air bubbles in the capillary. can also be caused by crystals in the polymer and/or fluorescent material in the polymer or formamide. unlike other artifacts, are generally seen in the same position in all colors.
Extreneous Peaks: Noise
non-reproducible background peaks that occur along the baseline in all samples. A wide variety of factors, including amplified current fluctuations within the electronic circuitry, air bubbles, urea crystals, and sample contamination, can create noise. If large enough (close to the laboratory threshold), they may be confused with an allele or mask alleles.
A human cell contains two types of DNA:
nuclear and mitochondrial.
A _____ is composed of a sugar, a phosphorous-containing group, and a nitrogen-containing molecule called a base.
nucleotide
The building blocks of the DNA molecule are known as:
nucleotides.
The Hardy-Weinberg principle:
predicts how gene frequencies will be inherited from generation to generation given a specific set of assumptions. states that in a large randomly breeding population, allelic frequencies will remain the same from generation to generation assuming that there is no mutation, gene migration, selection or genetic drift. can be illustrated mathematically with the equation: p2+2pq+q2 = 1 (Where 'p' and 'q' represent the frequencies of alleles. It is important to note that p added to q always equals one 100%.)
Clotting:
protein in plasma, fibrin, traps and enmeshes the red blood cells.
PSA (p30) is a:
protein unique to seminal plasma.
CODIS is a national system of:
shared databases of DNA typing information from convicted felons and crime scene evidence.
Extreneous Peaks: Pull up
sometimes referred to as bleed-through, represents a failure of the analysis software to discriminate between the different dye colors used during the generation of the data. Oversaturated data can also cause the dyes to "bleed" over or pull-up into another color.
Buccal cells are obtained from:
the mouth and inside of the cheek.
The skeletonized perimeter of a bloodstain can be used to interpret:
the time that elapsed between deposition of the stain and alteration of the stain.
A heterozygous locus will show two peaks with roughly equal allele heights.
true.
Which nitrogenous base is NOT found in DNA?
uracil (its RNA)
Presumptive testing chemicals used:
Tetramethylbenzidine (TMB) Tetramethylbenzidine is a mutagen and an irritant; avoid contact with metals. Glacial acetic acid is corrosive, combustible, and causes severe burns. Ethanol is highly flammable. Phenolphthalin (PHT) Phenolphthalin is an irritant. Phenolphthalein may be carcinogenic and may cause reproductive disorders. Potassium hydroxide is corrosive. Ethanol is highly flammable. Avoid contact with or inhalation of powdered zinc. Leucomalachite green (LMG) Leucomalachite green is an irritant; avoid contact or inhalation. Glacial acetic acid is corrosive, combustible, and causes severe burns. Luminol: In the case of luminol, the catalytic oxidation of the substrate compound produces light. Because the reagents are applied as a spray over a large area, the test is primarily used in the visualization of bloodstain patterns. The test is sensitive to at least 100 ppm and perhaps to 0.1 ppm of blood but will produce a false positive reaction in the presence of any of a number of oxidizing substances. Should a positive reaction take place, the result only suggests the presence of occult blood; the stain is therefore tested further with another presumptive test prior to reporting.
PCR Inhibitors:
The PCR process can be affected by compounds that interfere with the interaction between DNA and Taq polymerase, and thus inhibit the reaction. Many body fluids contain substances that can inhibit PCR.
Testing for seminal stains:
The best way to locate and at the same time characterize a seminal stain is to perform the acid phosphatase (an enzyme secreted into seminal fluid) color test. A purple color indicates acid phosphatase enzyme. Semen can be unequivocally identified by either the presence of spermatozoa or of p30, a protein unique to seminal plasma. Forensic scientists can successfully link seminal material to an individual by DNA typing.
Y-STR markers are useful when multiple males are involved in a sexual assault. If three men are involved in such an attack the investigators would expect Y-STR analysis to show a maximum of:
three peaks.
Mitochondrial DNA (mtDNA):
is located outside the cell's nucleus and is inherited from the mother. Mitochondria are structures found in all our cells used to provide energy that our bodies need to function. A single mitochondria contains several loops of DNA.
RFLP:
(Long process, requires high quality DNA, and lots of it) Length differences associated with relatively long repeating DNA strands are called restriction fragment length polymorphisms (RFLP) and form the basis for one of the first DNA typing procedures. Typically, a core sequence consists of 15 to 35 bases in length and repeats itself up to a thousand times. The key to understanding DNA typing lies in the knowledge that numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand. Once the DNA molecules have been cut up by a restriction enzyme, the resulting fragments are sorted out by electrophoresis. The smaller DNA fragments will move at a faster rate on the gel plate than the larger ones. The fragments are then transferred to a nylon membrane in a process called Southern blotting. To visualize the RFLPs, the nylon sheet is treated with radioactive probes containing a base sequence complementary to the RFLPs being identified (a process called hybridization). Next, the nylon sheet is placed against X-ray film and exposed for several days. When the film is processed, bands appear where radioactive probes stuck to fragments on the nylon sheet. A typical DNA fragment pattern will show two bands (one RFLP from each chromosome). When comparing the DNA fragment patterns of two or more specimens, one merely looks for a match between the band sets. A high degree of discrimination can be achieved by using a number of different probes and combining their frequencies.
How nucleotides can be linked to form a DNA strand:
(S) designates the sugar component, which is joined with phosphate groups (P) to form the backbone of DNA. Projecting from the backbone are four bases: A, adenine; G, guanine; T, thymine; and C, cytosine.
Using the product rule, what is the total frequency of the previous profile, given the following frequencies for each locus: D3S1358 = 0.04453 vWA = 0.02725 D5S818 = 0.07534
0.0000914
Results from DNA tests:
A match between two DNA samples (e.g. a crime scene stain and the DNA of a suspect) can link a subject to a crime. A non-match may shift the focus away from a particular suspect. Eligible profiles are entered into CODIS. Statistics are calculated to give weight to the evidence. A match with at least 13 loci would be considered identity. An STR DNA profile might be found in 1 person out of a quadrillion people. The approximate world population is 7.0 billion.
Acid Phosphatase -AP Spot Test -Reaction
Acid Phosphatase cleaves the phosphate from α‐napthyl phosphate to form napthol Napthol and Brentamine Fast Blue B forms a purple azo dye
Prostate Specific Antigen (PSA) p30 and test:
Antigen made in the prostate gland Weighs 30,000 kD Liquefies semen and is instrumental in dissolving the cervical mucous cap for sperm entry ABAcard® p30 looks for a reaction with sample and control.
Extraneous Peaks:
Artifacts: Spurious peaks blobs, spikes, noise Stutter 3′-A nucleotide addition Pull-up
DNA Base pairs:
As a result, adenine pairs with thymine and guanine pairs with cytosine.
Random match probability:
Assesses whether or not the match between the DNA profile obtained from the evidence and the DNA profile obtained from the suspect occurs by chance (coincidence). Allele frequencies are calculated using data that assumes Hardy-Weinberg Equilibrium (HWE) and linkage equilibrium. Homozygote frequencies are determined by p2 and heterozygote frequencies by 2pq. The genotypes from each locus can be multiplied (i.e. the product rule) to obtain the frequency of the combined individual genotypes. The combined frequency across multiple loci (non-linked) can be calculated by using the product rule, multiplying the respective frequencies together.
CODIS:
CODIS (Combined DNA Index System) is a computer software program developed by the FBI that maintains local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and profiles of missing persons. Currently, U.S. crime laboratories have standardized on 13 STRs for entry into a national database (CODIS).
Presumptive testing:
Catalytic tests for blood are based on the peroxidase-like activity exhibited by the heme group of hemoglobin. A colorless reagent is oxidized in the presence of heme and hydrogen peroxide. A positive reaction results in a color change. Oxidant (hydrogen peroxide) oxidizes a colorless reagent to a colored reagent. Heme catalyzes this oxidation by cleaving an oxygen from hydrogen peroxide (H2O2). The test is exceedingly sensitive to minute traces of hemoglobin and its derivatives but will produce a false positive reaction in the presence of any of a number of oxidizing substances. Should a color reaction take place, the result only suggests the presence of blood; the test is therefore a presumptive test. A reaction prior to the addition of hydrogen peroxide may be indicative of a false positive reaction and would be documented as inconclusive. Appearance of a rapid color change is a presumptive positive result for blood. Phenolphthalin: colorless → bright pink Tetramethylbenzidine: colorless → blue-green Leucomalachite Green: colorless → blue-green Appearance of no rapid color change is a negative result for blood
False positives: Presumptive testing
Chemical oxidants Catalysts Plant peroxidases Vaginal Acid Phosphatase Fecal Material Plant matter Spermicides Some feminine hygiene products
Sperm Cell Morphology:
Composed of a head, midpiece and tail Head Acrosome cap -aids sperm entry to egg Houses nuclear material (DNA) About 4.5 μm long and 2.5 μm wide Midpiece Houses mitochondria -energy for sperm cell Tail Used for mobility, made of protein Long and fragile -first part to breakdown and easily detached About 40 μm long
Presumptive Tests in order by sensitivity:
Luminol (5-amino-2-3-dihydro-1, 4-phthalazinedione) - fluorescence - +++++ (probable carcinogen)Benzidine - Blue color - sensitivity:++++ (carcinogen) Tetra methyl benzidine - Blue color - sensitivity:+++ (probable carcinogen) o-tolidine - Dark green color - sensitivity:+++ (probable carcinogen) Phenolphthalein or Kastle Meyer test - Pink color - sensitivity:++ (relatively safe) Leucomalachite green - Green color - sensitivity:++ (relatively safe)
Extreneous Peaks: Dye Blob
Dye blobs - Disassociated primer dyes, more commonly referred to as dye blobs, are fairly common in STR analysis.
Population Genetics:
Every diploid cell has two alleles, one inherited from each parent. If an individual has two different alleles at a specific locus, the individual is heterozygous at that locus. If the two alleles are the same, the individual is homozygous. A gene pool is the unique set of alleles that could be found by analyzing the DNA from every member of a species or population. A large gene pool is often associated with considerable genetic diversity whereas a small gene pool is associated with poor genetic diversity and can lead to decreased fitness and an increased chance of extinction.
Y-STRs:
Forensic scientists can also type STRs located on the Y or male chromosome. Y-STRs prove useful when multiple males are involved in a sexual assault. For example an STR pattern arising from three males will have only three bands. Another tool available in the arsenal of the DNA analyst is the ability to type STRs located on the Y chromosome, which is male specific. More than 20 different Y-STR markers have been identified. Y-STRs will prove useful when multiple males are involved in a sexual assault. A Y-STR analysis will have only one band or peak, rather than the conventional STR which is derived from two chromosomes and has two bands or peaks. The Y-STR is therefore less complicated in appearance and interpretation.
What method is used to identify spermatozoa?
Microscopy
Blood typing:
More than 15 blood antigen systems have been identified, but the ABO and Rh systems are the most important. Type A has A antigens on his/her red blood cells Type B has B antigens Type AB has both A and B antigens Type O has neither A nor B antigens Rh factor is determined by the presence of the D antigen. People having the D antigen are Rh positive; those not having the antigen are Rh negative.
PCR Inhibitors: collections process
Inhibitors can be introduced in the collection and analysis processes or at the crime scene. The powder from car airbags can inhibit too.
Extreneous Peaks: Stutter
Is a by-product of the amplification of STR loci whereby a minor product one repeat smaller than the primary allele is generated. Typically, stutter is effected by: The repeat unit length (2 basepair repeats have higher stutter than 3 basepair, etc). The degree of homogeneity of repeats (the more homogenous, the higher the stutter). The length of the allele within a locus (the larger the alleles have higher stutter).
What is multiplexing?
It is a technique that stimutaneously detects more than one STR in a single DNA analysis. It is important to DNA profiling because the more STRs a scientist can characterize, the greater chance that they originated from the same person.
Which of the presumptive blood tests is considered the most sensitive?
Luminol
Antibodies are found:
in the blood serum.
Quantitative PCR (qPCR):
Optimal concentration of input DNA is in the range of 0.5ng - 2ng. Adding too much or too little DNA to the amplification reaction can result in problems in the analysis. Quantitative PCR (qPCR), sometimes referred to as real time PCR, is the most accurate, precise, and efficient method currently available for human DNA quantitation. It is an amplification process that detects and measures the accumulation of fluorescent dyes as the reaction progresses. Quantitative PCR monitors the increase in fluorescent signal throughout the PCR cycling process. The change in fluorescence is monitored between samples and standards so that a comparison can be made. Uses the same cycles as PCR but incorporates dyes into the new copies. By detecting the increase in amount of dye present in each PCR cycle, the amount of "amplifiable" DNA present in the original sample can be estimated.
Species Origin Determination:
Ouchterlony: Uses radial diffusion of antigen and antibody through agar gel. Blood can be identified as being of human origin by precipitin reactions with antisera specific for components of human blood. Usually this is an anti-human serum serum - that is, an antiserum to human serum. This is a test for human origin not for human blood, as serum constituents such as albumin and some globulins are found in the extra-vascular space. Cross-over Electrophoresis: Cross-over electrophoresis for species identification is conducted using agar at a pH of 8.6. Stain extracts are loaded into wells arranged in a line at the cathode end of the plate and the antiserum is loaded into wells at the anode end. During electrophoresis, the electric field drives the serum proteins towards the anode, but the IgG molecules, which are essentially neutral at this pH, are driven to the cathode by the process of electroendosmosis. The antigen-antibody precipitation occurs at the interface between the two rows of wells. HemaTrace This test is designed to be used as a presumptive test for human (primate) origin. This rapid immunochromatographic test offers extremely high sensitivity and specificity and is capable of detecting trace levels of human hemoglobin. The detection limit has been shown to be 0.05 μg/mL in 10 minutes. All primate bloods produce positive results. Positive results may be obtained from whole blood from the domestic ferret. Immunochromatographic Assays Identification of human hemoglobin protein Hexagon OBTI ABAcard HemaTrace Identification of human glycophorin A protein RSID-Blood
Survival Times of sperm:
Persistence of semen components: Vagina / Cervix Acid Phosphatase = up to 7 days postmortem Sperm = up to 6 days Prostate‐Specific Antigen p30 = up to 2 days post-coitus Mouth Sperm about six hours Acid Phosphatase = up to 36 hours PSA p30 - less due to water solubility Anal cavity Sperm = 20-24 hours Acid Phosphatase = up to 24 hours Prostate Specific Antigen p30 - less Dried Stain Sperm, Acid Phosphatase, PSA p30 -years if proper storage
DNA Replication: PCR
Polymerase chain reaction (PCR) is a technique for replicating small quantities of DNA or broken pieces of DNA found at a crime scene, outside a living cell. For the forensic scientist, PCR offers a distinct advantage in that it can amplify minute quantities of DNA many millions of times. First, the DNA is heated to separate it. Second, primers (short strands of DNA used to target specific regions of DNA for replication) are added, which hybridize with the strands. Third, DNA polymerase and free nucleotides (A, T, G, and C) are added to rebuild each of the separated strands. Now, this cycle is repeated 25 to 30 times. Within a few hours a short strand of DNA can be multiplied a billion fold. Actual Steps: Initial incubation (95oC) - activates the hot start Taq Denaturation (94oC) - separates the 2 DNA strands Annealing (59oC) - Taq polymerase attaches primers to flank the DNA regions of interest Extension (72oC) - d NTPs start at primer and create a complimentary DNA strand to the template Repeat for 28 cycles Final extension (60oC) - attaches A nucleotides at the 3' ends of the strands
Population Genetics: Statistics
Population databases allow for estimations of how rare or common a DNA profile may be in a particular population. The CODIS software, PopStats, has the following population databases: African American Asian Caucasian Hispanic Native American The allele frequencies in the populations were gathered from the CODIS 13 core short tandem repeat (STR) loci.
Presumptive vs Confirmatory testing:
Presumptive - Indicates a substance is present, non-specific Confirmatory - Confirms a substance is present, specific
STR (short tandem repeats):
STRs normally consist of repeating sequences of 3 to 7 bases in length, and the entire strand of an STR is also very short, less than 450 bases in length. This means that STRs are much less susceptible to degradation and may often be recovered from bodies or stains that have been subjected to extreme decomposition. Because of their shortness, STRs are ideal candidates for multiplication by PCR, thus overcoming the previously mentioned limited-sample-size problem often associated with crime-scene evidence. What makes STRs so attractive to forensic scientists is that hundreds of different types of STRs are found in human genes. The more STRs one can characterize, the smaller will be the percentage of the population from which a particular combination of STRs can emanate. This gives rise to the concept of multiplexing. Using the technology of PCR, one can simultaneously extract and amplify a combination of different STRs.
Secretor:
Secretor - Almost everyone has at least trace levels of antigen in their body secretions that correspond to their ABO blood type. However, about 80% of the population has very high levels of these antigens in body secretions. These persons are described as secretors. The A, B, and H antigens are polysaccharides. They are found on RBC surfaces as lipo-polysaccharides and in secretions as glycoproteins.
Why do sperm survive longer?
Sperm heads, very strong Designed to last to penetrate egg Proteins are water soluble, breakdown quicker Sperm tails are lost first Tails are fragile and break off Made of proteins, bacteria attack first
Choose the correct conclusion for the following profile comparison: Evidence: D3S158 (15,16); vWA (16,16); D5S818 (12,13) Suspect: D3S158 (15,16); vWA (16,16); D5S818 (12,13)
Suspect is included.
Confirmatory tests:
Takayama or hemochromogen test - ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals or pyridine ferroprotoporphyrin. Teichman reagent - consisting of a solution of potassium bromide, potassium chloride and potassium iodide in glacial acetic acid, is heated to react with hemoglobin. The reaction first converts the hemoglobin to hemin, and then the halides react with the hemin to form characteristic brownish-yellow rhomboid crystals. Microscopic Examination
Frequentist Approach: random match probability
The frequentist approach uses probabilities or genotype frequencies to address statistical questions. Coincidence probability or random match probability (RMP) and the probability of exclusion are each frequentist approaches that are applied by the forensic science community.
Likelihood ratio:
The likelihood ratio is the ratio of two probabilities of the same event under different hypotheses. Thus for events A and B, the probability of A given that B is true (hypothesis #1), divided by the probability of event A given that B is false (hypothesis #2) gives a likelihood ratio. The likelihood ratio is a ratio of probabilities, and the higher the ratio, the more likely it is that the first hypothesis is true. In forensic biology, likelihood ratios are usually constructed with the numerator being the probability of the evidence if the identified person is the source of the evidence, and the denominator being probability of the evidence if an unidentified person is the source of the evidence. The results can be interpreted as follows: LR < 1 — the genetic evidence has more support from the denominator hypothesis LR = 1 — the genetic evidence has equal support from both numerator and denominator hypotheses LR > 1 — the genetic evidence has more support from the numerator hypothesis LR = P(E/H1) / P(E/H0) P(E/H1) is the probability of the evidence given a presumed individual is the contributor. P(E/H0) is the probability of the evidence given the presumed individual is not the contributor of the evidence.
Peak Height Percentage:
The peak height percentage for two heterozygous peaks is the peak height of the smaller peak divided by the peak height of the larger peak. Under optimum conditions (sufficient quantity and quality of DNA), heterozygous peaks within a locus should be similar in height or intensity to each other. In general, heterozygous alleles have peak heights that are within 70% of each other.
DNA Analysis:
The picture shows that the person is heterozygous for this allele. Data produced in the separation and characterization of amplified DNA is displayed as peaks (capillary electrophoresis). The software program designates peaks in electropherograms by sizing and makes allele calls through size comparisons to an allelic ladder.
PSA and Acid Phosphatase location:
The prostate is the source of the enzyme acid phosphatase and the protein Prostate‐Specific Antigen (PSA), or p30 protein
Hematrace:
This test is designed to be used as a presumptive test for human (primate) origin. This rapid immunochromatographic test offers extremely high sensitivity and specificity and is capable of detecting trace levels of human hemoglobin. The detection limit has been shown to be 0.05 μg/mL in 10 minutes. All primate bloods produce positive results. Positive results may be obtained from whole blood from the domestic ferret.
To determine whether a bloodstain is of human or animal origin, the serologist will perform:
a precipitin test.
The removal of an object or surface that was located between the origin of blood and the target surface during the bloodstain deposition leaves behind:
a void.
Four types of bases are associated with the DNA structure:
adenine (A), guanine (G), cytosine (C), and thymine (T).
The clumping together of red blood cells by the action of an antibody is known as:
agglutination.
Which of the following can give a false positive reaction with a presumptive blood test?
all of the above.
A stain can tentatively be identified as blood by:
all of these.
Evidence to substantiate that a rape occurred could include:
all of these.
Which is an advantage of working with short DNA fragments?
all of these.
An _________ is any of several alternative forms of genes at a particular locus and that are aligned with one another on a chromosome pair.
allele
The intersection of straight lines through the long axis of several individual bloodstains in an impact spatter pattern illustrates the _____ of the pattern.
area of convergence
The pressure of the pumping of oxygenated blood out of an injury causes bright red-colored blood to spurt out and form an:
arterial spray pattern.
A blood droplet deposited at an angle of impact about 90' (directly vertical to the surface) will:
be approximately circular in shape.
A trail pattern leading away from the victim at a stabbing scene was most likely created by:
blood dripping from the murder weapon.
Type AB blood contains:
both A and B antigens.
An expirated blood pattern at a crime scene is identified by:
bubbles of oxygen within the pattern.
By counting and pairing the patterns of a _____ pattern, an investigator can estimate the minimum number of blows inflicted upon a victim.
cast-off
Agglutination:
clumping together of red blood cells by the action of an antibody.
Luminol can be used at crime scenes to:
detect traces of blood.
In the PCR process, the first step is to heat the DNA strands. This is to permit:
double-stranded molecules to separate completely.
The approximate drying time of a pool of blood can be used to estimate timing of events at a crime scene and is dependent upon:
environmental condition of the scene.
The luminol test is specific for identification of blood
false
Means to detect the amelogenin gene are included in commercial STR kits used in crime labs because the gene allows determination of:
gender.
A _____ is the fundamental unit of heredity.
gene
The amount of acid phosphatase in seminal fluid is _____the amount of acid phosphatase in blood.
greater than
A ____ gene pair is made up of two different alleles
heterozygous
A ____ gene pair is made up of two similar alleles.
homozygous
The pointed end of a bloodstain always faces:
in its direction of travel.
