Introduction to body fluids Flashcards
Semen
- Often associated with homicides and other sexual offences.
- The typical ejaculate contains between 1.5 and 5ml of semen and contains between 40 and 250 million spermatozoa
- Nucleic DNA recovery is often undertaken from the sperm head.
- Rich source of DNA
Fluorescence
- Both saliva and semen fluoresce by using ultraviolet, alternative light sources
- In order to identify the presence of semen, commercially available semen detection kits are available.
- For the most part, these rely upon detecting the enzyme acid phosphatase on stained clothing.
- AP test is one of a number of presumptive or indicative tests which must be followed up with subsequent DNA analysis.
Draw back of the Acid Phosphatase test?
AP is a relatively commonplace enzyme and can often lead to a false positive result. In particular, onion skin and teabags can give a false positive AP result
What is Acid Phosphatase used for?
It is a presumptive test for semen
Is DNA found in red or white blood cells?
White cells which there are less of
how to preserve blood samples?
- For a number of forensic tests, we need to keep the blood sample whole and hence store the samples in vials containing EDTA.
- Blood samples taken requiring DNA will require to be preserved in EDTA.
Types of human blood cells
- White blood cells (Leucocytes) - they possess a nucleolus
- Red blood cells (Erythrocytes) and platelets - thoses which don’t possess a nucleolus
Percentage of red blood cells
45%
Percentage of white blood cells
1%
Faeces
- Faeces and urine are occasionally found at crime scenes due to the offenders wish to damage or violates the property or victim further.
- The new DNA 17 technique is showing signs of substantially better identification and discrimination of DNA.
- Historically the recovery of human DNA from faeces has been difficult due to the inhibitors which interfered with the standard (SGM Plus) test.
- Previously mitochondrial DNA was more closely associated with the examination of faeces but the new multiplexes will be better at extracting DNA from faecal material.
What happens when a presumptive test is negative?
- Analysis would cease at this point.
- There is little point in undertaking further testing where negative presumption has been made.
What happens if a presumptive test is positive?
It would go on for further confirmatory testing.
Luminol
It identifies where a material could be and is used for visualisation
Kastle Mayers test
Chemicals
- Ethanol
- Phenophalein
- Hydrogen peroxide
Saliva
- Product of exocrine glands (as is semen)
- They are secretions that are released to the outside of the body through ducts
- Saliva sampling is now commonplace in many forensic cases and it is conventional to take a number of swabs where the victim has thought to be bitten, kissed or sucked
- Likewise it is very common nowadays for DNA to be obtained from bite marks and spit; in particular in cases of sexual assault and murder
- The DNA 17 techniques will make these examinations even more relevant and productive
Elisa test
- Most common test for the presence of saliva staining.
- The same technique is also used to determine the presence of drugs and drug screening.
Phadebas
- Phadebas has a range of applications including the detection of saliva stains in forensic investigation and is now commonly used in laboratories.
- This test is capable of finding saliva deposits on various substrates.
- Fluoresses blue if saliva is present.
What is DNA?
Deoxyribonucleic Acid (DNA) is a molecule that encodes an organism’s genetic blueprint.
DNA is a ? formed of a collection of?
Building blocks of life?
Amino acids make up?
Proteins make up?
Cells make up?
- DNA is a moleculed formed of a collection of atoms.
- Building blocks of life are amino acids.
- Amino acids make up proteins
- Proteins make up cells.
- Cells make up tissue.
Functions of DNA
- DNA directs the machinery of a cell to make specific proteins.
- DNA stores the hereditary information of an individual.
- DNA can do complementary base pairing.
- DNA has the ability to mutate or change.
- This change occurs in a DNA sequence.
- Mutations are relatively common in our DNA, but most have no detectable effect.
- This mutation allows for new characteristics and abilities to appear which may help an individual to survive and reproduce (This is what we call EVOLUTION)
Introns
- Non coding areas of DNA that we’re interested in.
- The coding genes are the ones that we all share together.
DNA vs RNA
Key differences
- Structure RNA is usually single-stranded, while DNA is typically double-stranded in a helical form.
- Sugar RNA contains ribose sugar, while DNA contains deoxyribose sugar.
- Bases RNA uses uracil (U) instead of thymine (T) found in DNA. So, RNA has adenine (A), cytosine (C), guanine (G), and uracil (U), while DNA has adenine (A), cytosine (C), guanine (G), and thymine (T).
- Function DNA stores genetic information, while RNA is involved in protein synthesis, gene regulation, and other cellular functions.
- Location DNA is primarily located in the cell nucleus, while RNA is found both in the nucleus and the cytoplasm.
DNA Typing / Profiling
- Method of determining an individual’s DNA characteristics.
- Use on multiple sites of the chromosome because they’re polymorphic - lots of variations which makes the evidence stronger.
- Need to do population statistics to provide numerical values for the likelihood of the presence of certain chromosomes.
DNA Profiling Steps
4 basic steps
1) Purify the sample & Extract DNA
Extract from cells and separate from cellular components.
2) Use PCR to make copies of DNA.
3) 3) STR Analysis of DNA fragments - Use Gel Electrophoresis to separate them.
4) fter undergoing the above steps, a suspect’s DNA can be compared to DNA left at a crime scene.
DNA profiling
- Obtain a blood sample
- Obtain DNA from white blood cells.
- Mix this with restriction endonucleases to obtain DNA fragments.
- DNA fragments are loaded into agarose gel and separated by electrophoresis.
- DNA bands transferred to nylon membrane, which is then incubated with radioactive probes (complementary DNA with radioactive nucleotides)
- X-ray photographic sheet placed over membrane and later developed to reveal the series of DNA bands.
DNA Extraction Steps
- DNA Extraction removes DNA out of the nucleus and purifies it for analysis.
1. Lysis
2. Precipitation
3. Purification
DNA Extraction
Lysis
- The first step is to break open the cell and nucleus to free DNA.
- This is done by physically breaking it with a blender or mortar and pestle.
- Next, detergents are added to dissolve proteins to free the DNA.
DNA Extraction
Precipitation
Now sodium ions neutralize the negatively charged DNA and then alcohol is added to precipitate the DNA (form a solid).
DNA Extraction
Purification
- The DNA precipitate is washed with alcohol to remove any impurities.
- It is usually dissolved in water again for storage.
What is PCR?
- Polymerase Chain Reaction
- A method of quickly making millions of copies of a DNA sample for forensic analysis.
- Extremely valuable for small DNA samples found at crime scenes.
PCR Procedure
1) Heat the DNA strands so they unzip. Helicase unzipes the DNA and forms it into protein molecules
2) Cool and add a primer. The primer adds a complementary strand of DNA.
3) Add DNA polymerase and free nucleotides
Heat again for completion.
Short Tandem Repeats (STR)
- An area of nuclear DNA where there is repeating sequences, typically 4 bases, though 3 and 5 are also frequent.
- STR analysis compares repeats at specific loci in DNA.
- Unrelated individuals will have different numbers of repeating units.
- CODIS & NDIS work by looking at 13-17 different loci.
- The probability of two non-twin individuals with the same STR in 13 different locations is 1 in a quintillion.
- Identical twins will have identical DNA, therefore they will match on STR analysis
They must be differentiated with fingerprints.
Gel electrophoresis
- Method of separating DNA fragments by size and reactivity.
- DNA has a negative charge due to the sugar-phosphate backbone.
- In Electrophoresis, samples are put in the gel and an electric field is applied.
- The DNA samples migrate to the positive end of the gel.
- Longer DNA samples migrate a shorter distance; shorter DNA samples go further.
- Ethidium bromide is added as a dye as it will fluorescent under UV light.
Describe the process of DNA replication.
DNA replication is the process by which a cell makes an identical copy of its DNA. It involves the unwinding of the DNA double helix, the synthesis of new complementary strands by DNA polymerase, and the proofreading and repair of any errors.
What is the role of DNA polymerase in DNA replication?
DNA polymerase is the enzyme responsible for synthesizing new DNA strands during replication. It adds nucleotides to the growing DNA chain in a complementary fashion, using the existing DNA strand as a template.
How do you read a DNA profile?
Matching a suspect to crime DNA
- All suspects’ DNA is compared to the Crime Scene DNA.
- The suspect who has the same bands in the same location is the person who left the crime scene DNA.
What is a DNA profile?
- A DNA profile is a distinctive pattern of a person’s DNA (STRs) that have been separated by electrophoresis.
- Each person has a unique pattern except identical twins.
How to use a DNA profile to determone paternity?
- A person gets their DNA from their parents.
- DNA is compared to a known parent (mother) and which bands are not inherited from her had to come from dad.
Electropherogram
- In a complete profile, each person will exhibit either one or two peaks (alleles) at each gene locus.
- Loci that display only one allele indicate that the individual inherited the same marker from both parents at this locus.
- Where two alleles are displayed, the individual inherited different markers
- Issues arise when there may be more than one sample of DNA collected or the DNA may be degraded.
What is an electropherogram?
Graph produced when electrophoresis is used in DNA typing; shows the genetic material at each locus.
Increasing the themocycler will do what to DNA?
Denature
Source level DNA
Tells us if the DNA profile matches the swab.
Activity level DNA
How did the DNA get there
Offence
Hierachy of propositions
- Whether the person is guilty or innocent.
- For example the person of interest murdered the complaintant.
Activity
Hierachy of propositions
- An activity
- For example the person of interest had intercourse with the complainant.
Source
Hierachy of propositions
- Source of biological material
- For example the semen came from the person of interest.
Sub-source
Hierachy of propositions
- Source of DNA
- The DNA came from the person of interest.
Sub sub source
Hierachy of propositions
- Source of a specified component of the DNA profile ( eg major or minor component)
- The major DNA component came from the person of interest.
R v David Butler
- In 2005, Anne Marie Foy was beaten and strangled to death in Liverpool, UK. David Butler, a taxi driver was charged with her homicide after a 2010 database match to DNA from fingernail clippings collected from Ms. Foy. The sub-source LR was reported as one-thousand billion.
- Mr. Butler denied all involvement. He was known to have a condition that resulted in excessively flaky skin and his defence lawyer argued that the DNA recovered from Ms. Foy’s fingernails was transferred via secondary contact. Mr. Butler was convicted and subsequently acquitted after eight months in jail. Mr. Butler was quoted as saying, “…They thought it was my DNA, ergo it must be me.”
- This case was used to argue that DNA evidence is “flawed”.
R v David Butler
Error
- DNA profiling technology is not flawed.
- The error in this case was incorrectly assigning a sub-source LR reported for a match to DNA recovered from fingernail clippings to an activity level proposition.
Partial copies of DNA, shorter & containing only one side of the ladder?
Proteins which read the RNA code, three letters at a time?
DNA precipitate is washed with alcohol in the step
EPG =
The peaks on the EPG represent positions on the?
DNA is a …… charged molecule?
- RNA is partial copies of DNA, shorter & containing only one side of the ladder.
- Proteins which read the RNA code, three letters at a time are known as ribosomes.
- DNA precipitate is washed with alcohol in the purification step.
- EPG = Electropherogram
- The peaks on the EPG represent positions on the chromosome.
- DNA is a negatively charged molecule?
