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
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Introduction39
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Practical DNA Processing39
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More on the Practical Aspects of DNA Profiling33
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Benefits of Modern DNA Multiplexes24
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Interpretation of a Single Source Unmixed Profile18
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Contamination26
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Mixtures53
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Artefacts and Noise42
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Forensic Investigative Genetic Genealogy29
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Next Generation Sequencing30
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DNA Structure30
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DNA Stability and Handling23
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Electrophoresis14
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Synthesis22
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PCR29
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Profiling and Sequencing11
