Introduction Flashcards
What are the two preparation methods for bone samples?
- cryogenic method
- mortar and pestle
What is cryogenic method of preparing bone samples?
- freeze bone sample in liquid nitrogen which makes bone tissue brittle
- once frozen, the bone is crushed into a fine powder in freezer mill using mortar and pestle
- pulverised bone then subjected to further steps to extract DNA
What is mortar and pestle method of preparing bone samples?
- bone sample is manually ground into a fine powder using mortar and pestle
- physical grinding breaks down bone structure to release DNA
- subjected to further steps to extract DNA
What is the 8 step process of DNA extraction from bone sample
1 - sample preparation
- obtain a bone sample and clean the exterior surface to remove any potential contaminants using abrasion
- immersed in a bleach/detergent solution (to physically removing the outer bone surface) or exposed to UV radiation
- if necessary break down bone sample using saw or other suitable tools
2 - decalcification
- if bone sample is hard or mineralised, decalcify it using a decalcifying solution to remove calcium ions that can inhibit DNA extraction
3 - disruption of bone tissue
- either method to grind bone sample into fine powder
- this helps expose DNA trapped within bone tissue
- increases SA to various chemicals in DNA extraction to get more DNA
4 - cell lysis
- add a lysis buffer containing detergents and enzymes to break down cell membranes and release DNA from cells
- incubate sample at an appropriate temperature to ensure complete cell lysis
5 - protein digestion
- treat the lysed sample with proteinase K to digest proteins and remove them from the DNA solution
- incubate the sample to allow protein digestion to occur effectively
6 - DNA extraction
- perform DNA extraction using a method such as phenol-chloroform extraction, silica column-based extraction, or Chelex resin extraction
- follow the specific protocol of the chosen extraction method to isolate DNA from the sample
- centrifuge the sample to separate DNA from other cellular components
7 - DNA purification
- wash the extracted DNA to remove any remaining contaminants or impurities
- elute the purified DNA in an appropriate buffer for downstream applications
8 - quantification and quality check
- measure the concentration of the extracted DNA using a spectrophotometer or fluorometer
- assess the quality of the DNA by analysing its purity and integrity through methods like agarose gel/capillary electrophoresis
What are the advantages of cryogenic method over mortar and pestle method?
1 - preservation of DNA integrity
- cryogenic freezing in liquid nitrogen helps preserve the integrity of DNA by minimising degradation that can occur during mechanical grinding processes
2 - efficient cell disruption
- freezing the bone sample makes it brittle, facilitating easier and more efficient cell disruption when compared to manual grinding with a mortar and pestle.
3 - reduced contamination
- cryogenic grinding minimises the risk of contamination as the sample is less likely to eb exposed to external contaminants during the grinding process
4 - consistency in particle size
- cryogenic grinding tends to produce a more uniform and fine powder, which can lead to better DNA extraction yields and quality
What are the advantages of mortar and pestle method over cryogenic milling?
1 - cost-effective
- the mortar and pestle method is generally more cost effective and accessible as it doesn’t require specialised equipment like liquid nitrogen
2 - simple and traditional
- this method is straightforward and has been used for a long time in laboratories making it a familiar technique for many researchers
3 - suitable for small samples
- m+p method can be more suitable for processing small bone samples where cryogenic methods may not be as practical
What does choice between two methods depend on?
- depends on:
- scale of study
- available resources
- specific requirements of DNA extraction process
Why do we decalcify bone samples before DNA recovery?
1 - facilitates cell lysis
- calcium ions in bone tissue can hinder cell lysis process
- decalcifying means calcium ions are removed
2 - prevents DNA degradation
- calcium ions can promote DNA degradation by nucleases that are released during cell lysis
- removing calcium ions reduces the risk of DNA damage and degradation ensuring better quality DNA recovery
3 - improves efficiency
- decalcification helps in breaking down hard and mineralised bone tissue making it easer to grind the bone sample into a fine powder for subsequent extraction steps
- improves overall efficiency of DNA recovery from bone sample
4 - enhances purity
- decalcification helps in removing mineral components from bone sample reducing potential contaminants that could interfere with DNA extraction and purification processes
- more purer DNA sample for downstream applications
5 - prevents interference
- calcium ions can interfere with enzymatic reactions involved in DNA extraction e.g. action of proteinase K during cell lysis
- removing calcium ions ensures these enzymatic reactions proceed smoothly leading to successful DNA recovery
- better quality and higher yield of DNA for analysis
Fluorescence:
- what is it used for?
- saliva and semen fluoresce by using UV light sources
Semen:
- where is it produced?
- what type of DNA recovery is often undertaken from sperm head
- what can be a problem with sexual offenders in forensic science
- what is most encountered technique in semen analysis? what used to be?
- production of exocrine glands
- nucleic DNA recovery is often undertaken from sperm head
- some people will fail to ejaculate/ had a vasectomy or suffer from a medical condition that their semen lacks spermatozoa (azoospermia (no sperm), oligospermia (low sperm count))
- laser micro detection
- differential extraction method of sperm and vaginal epithelial cells from vaginal fluid mixed with semen used to be most encountered technique
Blood:
- how must blood samples be stored?
- what are two categories of human blood cells?
- what is composition of blood in terms of RBC and WBC
- how can bloodstains be visualised
- we need to keep blood sample whole and hence store samples in vials containing EDTA (ethylenediaminetetraacetic acid) - an anticoagulant
- those with nucleus (leucocytes/WBCs)
- those without (RBC, erythrocytes, platelets)
- blood is composed of 45 % RBC and 1 % WBC by volume
- infrared imaging can be used to demonstrate the presence of latent bloodstains without use of chemicals
Faeces:
- when are they found at crime scenes
- why did recovery of human DNA from faeces used to be difficult
- why is it not anymore
- what DNA is used (what did it used to be)
- when offender wants to damage/violates property or victim further
- recovery of human DNA from faeces has been difficult due to inhibitors which interfered with the standard (SGM plus) test
- new DNA 17 test shows better identification and discrimination of DNA
- new multiplexes will be better at extracting DNA
- previously mitochondrial DNA was more closely associated with examination of faeces
Saliva:
- where is it found?
- where are saliva swabs taken?
- production of exocrine glands
- take swabs where victim is thought to be bitten, kissed, and sucked
What can be said about positive and negative results from presumptive testing?
- positive - presumption so need further confirmatory testing
- negative - rejection and analysis would cease
AP test
- what does it test for?
- what is method?
- why can this test be a problem
- what type of test is it and what does this mean
- tests for acid phosphatase (semen)
- area is wetted and a sheet of filter paper is placed over area
- reagent sprayed
- commercial detection kids rely on detecting acid phosphatase (AP) enzyme on stained clothing
- AP is a common enzyme so can give false positives
- can also test positive for vaginal discharge so only used as early screen (followed by sperm head presence test)
- it is a presumptive/indicative test which must be followed up with subsequent DNA analysis
Luminol presumptive test:
- what does it test for
- what is positive test
- presumptive test for blood
- positive test: emission of light
Kastle-Meyer presumptive test:
- what does it test for?
- what is process?
- what is positive result?
- how can interference with subsequent DNA analysis be avoided?
- presumptive test for blood
1 - filter paper is rubbed on stain
2 - add drop of ethanol to filter paper (improves sensitivity)
3 - add drop of phenolphthalein to filter paper
(if paper turns purple now = contaminated result)
4 - hydrogen peroxide is added
- positive result: pink reaction on filter paper (H2O2 interacts with haem = pink)
- by applying chemicals to filter paper and not garment or surface and test
LMG presumptive test:
- what does it test for?
- what is process?
- what is positive result?
- presumptive test for blood
1 - filter paper is rubbed on stain
2 - add ethanol
3 - add LMG solution
4 - add hydrogen peroxide
- positive result: blue/green colour change
Confirmatory test:
- used to be?
- used to do what?
- process
- what is new way
- used to be precipitin test
- used to identify if it is human or animal blood
1 - animal is injected with human blood
2 - animal makes anti-human antibodies
3 - animal serum is extracted
4 - when human blood added to this you get precipitin band
- nowadays would go straight for DNA
Phadebas:
- what does it test for?
- when useful
- how does it work
- positive test?
- presumptive test for saliva (replaced ELISA)
- useful when saliva is not present
- region of possible saliva staining is wetted and swabbed with cotton swab
- swab is wrapped in Phadebas paper (paper is imprinted with starch and blue dye) and placed under a weight
- will fluoresce blue where saliva is (amylase in saliva digests starch in paper)
ELISA test:
- name
- what is it test for
- enzyme-linked immunosorbent assay
- presumptive test for saliva
What are the four functions of DNA?
1 - it directs the machinery of a cell to make specific proteins
2 - it stores the hereditary information of an individual
- coding area (exon) - 98 % is shared between everyone
- non-coding area (intron) - found on chromosomes
3 - has the ability to mutate
- most have no detectable effect
- mutation allows for new characteristics and abilities to appear which may help an individual to survive and reproduce (evolution)
4 - complementary base pairing (AT and GC)
DNA vs RNA:
- structure
- sugar
- bases
- function
- location
DNA
- double stranded in helical form
- contains deoxyribose sugar
- ACGT
- stores genetic information
- located in cell nucleus
RNA
- single stranded
- contains ribose sugar
- ACGU
- involved in protein synthesis, gene regulation and other cellular functions
- nucleus and cytoplasm
What is DNA profiling?
- also known as DNA typing
- method of determining an individuals DNA characteristics using the non-coding parts of DNA
1 - purify the sample and extract DNA (extract from cells and separate from cellular components)
2 - quantify amount of material recovered
3 - use PCR to make copies of DNA
4 - STR analysis of DNA fragments (use gel electrophoresis to separate them)
5 - can then compare suspects DNA to DNA left at crime scene
STR:
- name
- what are they
- what does STR analysis involve
- what about identical twins
- short tandem repeats
- an area of nuclear DNA where there is repeating sequences (typically 4 bases)
- STR analysis compares repeats at specific loci in DNA (17)
- unrelated individuals will have different numbers of repeating units
- identical twins will have identical DNA so they will match on STR analysis
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
DNA profile:
- what is it
- how are they compared
- where is DNA inherited from
- distinctive pattern of a persons STRs that have been separated by electrophoresis
- look at bands in same location when matching suspect to crime scene DNA
- a person gets their DNA from their parents (half from mother and half from father)
Electropherogram:
- what is it
- what does it show
- homozygous or heterozygous?
- what can be issues?
- it is the graph produced when electrophoresis is used in DNA typing
- it shows the alleles present at each gene locus
- peaks on EPG represent positions on chromosome
- homozygous = 1 allele at loci (2 identical alleles)
- inherited same marker from both parents
- peak is usually taller
- heterozygous = 2 alleles at loci
- inherited from one parent
- issues:
- when more than one sample of DNA collected
- DNA may be degraded
What is the hierarchy of propositions?
- offence (guilt or innocence)
- activity (an activity)
- source (source of biological material)
- sub-source (source of DNA)
- sub-sub-source (source of a specified component of DNA profile)
R v David Butler case
- summarise
- error
- in 2005, Anne Marie Foy was beaten and strangled to death in Liverpool
- David Butler (taxi driver) was charged with her homicide - found match to DNA from fingernail clippings of Anne
- sub-source likelihood ratio was reported as 1-thousand billion
- Butler denied involvement
- he had condition (flaky skin) so lawyer argued secondary contact
- Butler was convicted falsely due to DNA match
- ERROR:
- incorrectly assigned a sub-source LR reported for a match to DNA recovered from fingernail clippings to an activity level proposition (strangulation in this case)
Maria Marchese
- she put DNA evidence from male from condom into her underwear and claimed she was SA
- DNA evidence was dismissed as found lubricants and spermicides within DNA
What can be said about viewing DNA findings
- DNA findings have to be viewed within the context of both the prosecution and defence hypothesis
What are the proteins called which read the RNA code three letters at a time?
- ribosomes
What type of evidence can DNA evidence provide?
- exculpatory
What enzyme synthesises RNA primers?
- RNA primase
What glands secrete substances by the way of a duct?
- exocrine
What are red blood cells called?
- erythrocytes
What is maternally inherited DNA
- mitochondrial
