Practical DNA Processing Flashcards
1
Q
Equipment used to collect DNA from bone samples?
Detergents are added to dissolve proteins, to free DNA in this step
We refer to a single peak on an EPG as?
DNA came from the POI addresses this level of proposition?
Guanine and cytosine are complementary base pairs – true or false?
The structure of DNA, what attach to the sugar/phosphate backbone?
A
- Equipment used to collect DNA from bone samples is a freezer mill.
- Detergents are added to dissolve proteins, to free DNA in the lysis step.
- We refer to a single peak on an EPG as homozygous.
- DNA came from the POI addresses sub source level of proposition.
- Guanine and cytosine are complementary base pairs is true.
- The structure of DNA, bases attach to the sugar/phosphate backbone.
2
Q
Practical DNA processing basic steps
A
- Firstly, steps against possible contamination. Before anyone enters the lab, they must be wearing personal protective equipment.
- Any items which are brought into laboratory are first wiped down with alcohol.
- The next step is sample retrieval – all evidence which comes into the lab must first be recorded, photographed and drawn with annotations before any samples are taken.
- This ensures a complete log exists of the item in question. If any damage is caused during subsequent testing this can be noted. It is a case of finding a potential source of DNA such as blood, saliva or other body fluid and then testing it in order to see whether or not it is in fact that substance.
3
Q
Sample retrieval
A
- All evidence which comes into the lab must first be recorded, photographed and drawn with annotations before any samples are taken.
- This ensures a complete log exists of the item in question.
- Check pockets, look for obvious staining and turn items inside out,
- Anything that looks like semen should be visualised, possibily with UV light.
- Saliva should be illuminated with phabedas.
- If any damage is caused during subsequent testing this can be noted.
- It is a case of finding a potential source of DNA such as blood, saliva or other body fluid and then testing it in order to see whether or not it is in fact that substance.
4
Q
Differential extraction
A
- Differential extraction is an important procedure where mixed DNA samples require separation of male and female genetic materials.
- The process hinges on the selective rupture of male cells, thereby isolating their DNA and facilitating the individual identification of each contributor within the mixed samples.
- This tactic rests on the fundamental understanding that the male genetic code comprises two copies of the X and Y chromosomes, contrasted with the female’s double-X chromosome makeup
- Extraction technique is used for sexual offence cases and is a two step process.
5
Q
LMD
A
Laser microdissection
6
Q
How does differential extraction work?
A
- Have a microscope with a laser attached
- We can look through the microscope and see where the sperm heads are to isolate them by cutting out that part of garment.
- You can be very specific.
7
Q
Supernatant
A
Liquid on top of centrifuged sample
8
Q
Cell lysis
A
The dissolution of structures such as sperm head or a cell so that the components of the structure go into free solution complete solubilisation (lysis).
9
Q
Real time PCR (qPCR)
A
- Real-time PCR aka quantitative PCR (qPCR), allows for the monitoring of amplification of a targeted DNA molecule during the PCR process.
- It provides real-time data on the amount of DNA present in a sample as the reaction progresses. This method is ideal for quantifying DNA and detecting the presence of specific sequences.
- Real-time PCR allows for the (1) quantification of DNA during the amplification process, while (2) standard PCR does not provide this real-time monitoring capability..
10
Q
Standard PCR
A
- Standard PCR, on the other hand, does not provide real-time monitoring of the DNA amplification process.
- Instead, the amplification is carried out for a set number of cycles, after which the products are analysed. Standard PCR is commonly used for DNA amplification in research, diagnostics, and forensics, but it is not suitable for real-time quantification of DNA.
11
Q
How is qPCR done?
A
- Real-time PCR, is performed by continuously monitoring the amplification of a targeted DNA molecule during the PCR process.
- Achieved by using fluorescent dyes or probes that emit signals as the DNA amplification occurs.
- The real-time data allows for the quantification of the initial amount of DNA in the sample, making it suitable for various applications. Essentially – we can skip the quant phase.
12
Q
1.qPCR removes the need for this process (carried out in standard PCR)
2. Method is ideal for quantifying DNA and detecting the presence of ?
3. Achieved by using?
4. In standard PCR, the amplification is carried out for a?
5. qPCR allows for the
A
- qPCR removes the need for quantification process that is carried out in standard PCR.
2. qPCR is ideal for quantifying DNA and detecting the presence of specific DNA sequences.
3. This is achieved by using fluorescent dyes or prones,
4. In standard PCR, the amplification is carried out for a set number of cycles between 31-32 (used to be 28).
5. qPCR allows for the quantification of the initial amount of DNA.
13
Q
What is the role of DNA polymerase in DNA replication?
A
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.
14
Q
Explain the significance of the leading and lagging strands in DNA replication.
A
- In DNA replication, the leading strand is synthesized continuously in the 5’ to 3’ direction, while the lagging strand is synthesized discontinuously in short fragments called Okazaki fragments. This is significant because the lagging strand requires additional steps, such as the synthesis of RNA primers and the joining of Okazaki fragments by DNA ligase.
- Lagging strand needs more work and goes in the opposite direction.
15
Q
DNA comparison
A
- The DNA profile is compared to profiles currently on the National DNA Database.
- These are ones from persons who are arrested for recordable offences.
- Profiles produced using earlier forms of DNA profiling cannot be matched to current records which are undertaken using the current processes.
16
Q
Steps against contamination
A
- All people who enter laboratory must provide elimination DNA samples so that all final results can be checked against the employee and visitor database.
- To reduce the risk of this happening, all staff must wear personal protective clothing.
17
Q
Order of PPE according to SOP
A
- Facemask
- Mobcap
- Nitrile gloves
- Lab coat
- Second pair of gloves
18
Q
Contamination measures
A
- Whilst alcohol will not, on its own, destroy DNA – the physical act of friction over the surfaces helps keep them free of unwanted materials.
- Gloves are wiped between each sample as are the benches and equipment.
- Each bench has its own set of equipment which is logged so – should contamination be found it can be traced back to the piece of equipment or the bench responsible.
19
Q
Sample retrieval log
A
- As an item is logged in, a note taken of the bench on which it will be examined on and the name of the scientist undertaking the examination.
- The laboratory takes his own photographs in order to show what condition the item was in when it arrived.
- If there is any damage caused during transit then it can be noted where any marks or tears occurred.
- Then the item’s description is logged as a written report.
20
Q
Drawings
A
- Are done in pen so that they cannot be easily changed later.
- No correction fluid at all is allowed on evidence logs.
- If a mistake is made then it is crossed through, initialed and dated to show that the correction is authorised and identifying when it happened.
- When an item has been recorded in such way, if the worst were to happen and the item were destroyed then it could still be referred to as evidence.
21
Q
Does the recovery of sequence matter?
A
- It does matter which order the tests are done. Essentially tests are done in order of the increasing likelihood of damage.
- Firstly, any hairs and fibres are removed so they don’t have any possibility of being destroyed or being lost.
- Then blood is tested using a dry swab. Any greasy stains are noted by eye and recorded.
- Then, any areas which they may be small samples of touch DNA are swabbed in case there is a low level of DNA.
- Wet tests are then performed. These are done last as any wetting of the exhibit may damage or destroy some evidence.
- The items are tested for saliva first and then for semen.
22
Q
Visable staining
A
- In the case of this pair of jeans the obvious red stains are tested for blood.
- This is done by taking the circle of filter paper, folding into quarters and using the point of the swab to rub against the stain. The filter paper is tested with 1 to 2 drops of presumptive testing solution.
- Either LMG or KM and assuming there is no colour change at this point (remember – if it changes colour on the first drop then the kit is most likely contaminated).
- 1 to 3 drops of hydrogen peroxide is then added and if any colour change noted (blue/purple) then the stain is possibly blood.
23
Q
Phadebas test
A
- In order to test for saliva , the process is referred to as the phadebas test.
- This is paper which is imprinted with (a) starch and contains a (b) blue dye.
- In order to do the test, the region of possible saliva staining is wetted and then swabbed with a cotton swab.
- The swab is then wrapped in phadebas paper and placed under a weight.
- Due to the amylase in the saliva which digests the starch the dye will run in the presence of saliva.
- If no saliva is present, even if the swab is wet the dye will not run.
- If a sample is highly sourced (has much saliva present) it may even bleach the paper.
24
Q
Non-visible staining
A
- Sometimes stains are not so obvious
- For example, searching for semen stains may require a dark room and alternative light sources.
- When semen, or other body fluids (or other organic material – for that matter) they may fluoresce under ultraviolet light when viewed through filtering goggles.
25
Q
AP test
A
- When a possible semen stain is found the area is wetted, a sheet of filter paper is placed over the area and the region of the testing is noted.
- The sheet is sprayed with a reagent as part of the acid phosphatase test.
- Luminscence indicates semen staining.
- The AP test can, for example test positive for vaginal discharge and is therefore (like other presumptive tests) only used as an early screen.
- For example, the definitive test cases of sexual offence is to note the presence of sperm head.
26
Q
Can you differentiate between male and female blood?
A
No
