Practical DNA Processing

Question 1

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?

Answer 1

• 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.

Question 2

Practical DNA processing basic steps

Answer 2

• 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.

Question 3

Sample retrieval

Answer 3

• 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.

Question 4

Differential extraction

Answer 4

• 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.

Question 5

LMD

Answer 5

Laser microdissection

Question 6

How does differential extraction work?

Answer 6

• 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.

Question 7

Supernatant

Answer 7

Liquid on top of centrifuged sample

Question 8

Cell lysis

Answer 8

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).

Question 9

Real time PCR (qPCR)

Answer 9

• 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..

Question 10

Standard PCR

Answer 10

• 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.

Question 11

How is qPCR done?

Answer 11

• 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.

Question 12

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

Answer 12

• 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.

Question 13

What is the role of DNA polymerase in DNA replication?

Answer 13

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.

Question 14

Explain the significance of the leading and lagging strands in DNA replication.

Answer 14

• 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.

Question 15

DNA comparison

Answer 15

• 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.

Question 16

Steps against contamination

Answer 16

• 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.

Question 17

Order of PPE according to SOP

Answer 17

• Facemask
• Mobcap
• Nitrile gloves
• Lab coat
• Second pair of gloves

Question 18

Contamination measures

Answer 18

• 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.

Question 19

Sample retrieval log

Answer 19

• 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.

Question 20

Drawings

Answer 20

• 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.

Question 21

Does the recovery of sequence matter?

Answer 21

• 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.

Question 22

Visable staining

Answer 22

• 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.

Question 23

Phadebas test

Answer 23

• 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.

Question 24

Non-visible staining

Answer 24

• 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.

Question 25

AP test

Answer 25

* 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.

Question 26

Can you differentiate between male and female blood?

Answer 26

No

Question 27

1. Any items brought into the laboratory are first wiped down with? 2. All evidence which comes into the laboratory must first be? 3. Process of taking a small sample of DNA and copying it many times? 4. Arranges fragments of DNA by their molecular weight 5. Or people who into the laboratory must provide? 6. All notes and drawings are completed in? 7. A gateway test for saliva? 8. Location test for possible semen staining

Answer 27

1. Any items brought into the laboratory are first wiped down with alcohol 2. All evidence which comes into the laboratory must first be recorded. 3. PCR is the process of taking a small sample of DNA and copying it many times. 4. Electrophoresis arranges fragments of DNA by their molecular weight. 5. People who into the laboratory must provide elimination samples. 6. All notes and drawings are completed in pen. 7. A gateway test for saliva is phadebas. 8. Location test for possible semen staining is acid phosphatase.

Question 28

Body fluid stain

Answer 28

- When the stain has been found to be a body fluid, it is noted on the drawing. - Different coloured pencils are used to note different body fluids. - For example, blood is noted in red and whilst each laboratory will have its own code (which colours represent which body fluids) semen will be noted in purple.

Question 29

Cell harvesting

Answer 29

* Now we have a piece of filter paper or cotton bud swab these are likely to contain body fluid. * The next step is to harvest the cells and extract the DNA from these cells. * The sample is wetted to hydrate it and then it is centrifuged so that the liquid rises to the top and pellet of cells will be at the bottom of the tube and can be removed. * As part of the DNA extraction for semen and saliva, the cells must first be harvested.

Question 30

DNA extraction

Answer 30

Removal of DNA from cellular material; isolation or purification of DNA.

Question 31

If a pellet of cells is suspected to be semen, what should be donr?

Answer 31

- If the body fluid was suspected to be semen then it would need to be confirmed beforehand. - The pellet would have a sample taken and examined under a microscope in order to see if any sperm heads are seen. - Other fluids would go straight to the later stages of DNA.

Question 32

Basic principles of DNA extraction

Answer 32

1. Disrupting the cells 2. Separating DNA from other components 3. And purifying it for further analysis

Question 33

Organic extraction

Answer 33

- Organic extraction involves the use of organic solvents to break down the cells and isolate the DNA. - Isolate DNA using organic solvents.

Question 34

Solid phase extraction

Answer 34

- Solid-phase extraction utilizes specialized columns or magnetic beads to selectively bind DNA.

Question 35

How is DNA precipitated?

Answer 35

Using alcohol

Question 36

Solid extraction of DNA

Answer 36

- This is useful samples where it is likely that they will contain low levels of DNA and so require an additional step. - This may be swabs of touch DNA or pieces of solid material such as paper, cigarettes or fabric or for swabs taken during the sample retrieval process. - The sample is placed into a piece of equipment and spun at high speed in order to release as much DNA from the swab or material as possible.

Question 37

Epithelial extraction

Answer 37

- Epithelial extraction deals with highly sourced samples - For example direct amounts of skin other tissue or other bodily fluids.

Question 38

Differential extraction of DNA

Answer 38

- Differential extraction is used for samples likely to contain sperm. - This extraction process consists of several washing steps are in order to remove any other sources of DNA such as female cells. - For example from underwear in a sexual offence case. This isolates the sperm.

Question 39

Cell lysis

Answer 39

- Where DNA is released into a solution by denaturing the cell wall, the enzyme degradation is prevented. - Proteinase K is used for the destruction of proteins in cell lysates. - ATL is a tissue lysis buffer used for purifcation. - Elution buffer is used to release DNA molecules that are bound the the solid phase Helps to separate target molecules

Question 40

DNA isolation process

Answer 40

This removes the unwanted cell debris from the solution and uses the reagent AL and then 100% ethanol is added as the DNA bind to silica column thus making it easier to extract.

Question 43

DNA purification process

Answer 43

- This is the last step in DNA extraction. - It is the release of DNA from the silica. - DNA is more stable at a slightly basic pH and will dissolve faster in the buffer than in water. - This is a process of removing impurities or inhibitors such as dyes from clothing, soil or coffee. - This is done by the addition if reagents AW1, AW2 (washing buffers) and by filtering. - This happens after an elution buffer is added. - This breaks down the hydrogen bonds between the DNA and the silica column, allowing the DNA to be released into a tube where it is centrifuged. - Filtering happens in a microcon which acts similar to a sieve, filtering the DNA and removing the inhibitors and impurities.

Question 44

1. DNA is released into a solution by doing what to the cell wall? 2. Extraction method used to isolate sperm? 3. Where the body fluid is suspected to be semen, we would need to confirm this by looking for? 4. In our notation, we would use this colour pencil to note the presence of blood? 5. Initiating the positive reaction in the KM presumptive test 6. We may try to locate semen by this method?

Answer 44

1. DNA is released into a solution by doing denaturing the cell wall? 2. Differential extraction method is used to isolate sperm. 3. Where the body fluid is suspected to be semen, we would need to confirm this by looking for sperm heads. 4. In our notation, we would use a red colour pencil to note the presence of blood. 5. Hydrogen peroxide initiates the positive reaction in the KM presumptive test. 6. We may try to locate semen by an alternative light source.

Question 45

The extraction negative

Answer 45

- A blank sample that is extracted using the same reagents and protocol as the samples on the extraction batch. - It is primarily used to identify contamination whether it be from potentially contaminated reagents or the cleanliness of the person/instruments performing the extraction. - So extraction negative which produces zero peaks suggest that the extraction has been successful and has not been affected by contamination.

Question 46

What are the key enzymes involved in DNA replication?

Answer 46

Several enzymes play crucial roles in DNA replication. These include DNA helicase (unwinds the double helix), DNA polymerase (synthesizes new DNA strands), DNA ligase (joins Okazaki fragments on the lagging strand) and RNA primase (synthesizes RNA primers).

Question 47

Discuss the importance of fidelity in DNA replication.

Answer 47

Fidelity in DNA replication refers to the accuracy with which DNA is copied. It is crucial for maintaining the genetic information without introducing errors. DNA polymerase has proofreading capabilities to correct any mistakes during replication, ensuring high fidelity in the process.

Question 48

Quantification of DNA

Answer 48

- Before the DNA can be replicated (so there is enough to sequence) it must first be quantified. - This allows the calculation of exactly how much DNA is present. It is vital to know this in order to prepare the sample for electrophoresis. - There are many main methods of quantitation which all work in a similar way by adding a fluorescent probe or dye to the sample and then comparing how much the sample fluorescence against control samples or standards already loaded in the equipment.

Question 49

Types of quantifier often used to quantify DNA?

Answer 49

- Quantfiler - PicoGreen HY

Question 50

Quantfiler

Answer 50

- Used to quantify DNA - Not human specific but quick and easy. - Advantageous because it gives an indication of how much male DNA is present.

Question 51

PicoGreen HY

Answer 51

A highly sensitive fluorescent nucleic acid stain which is used to quantitate double-stranded DNA.

Question 52

Denaturation of DNA

Answer 52

- This is the process of separating DNA - Most (in this laboratory) run for 96°C for five seconds although may differ slightly depending on the products used.

Question 53

Annealing of DNA

Answer 53

- The process of two strands of DNA rejoining. - 20°C for 35 seconds although may differ slightly depending upon the products used.

Question 54

Annealing of primers

Answer 54

- Small pieces of DNA that provide the free ends needed for DNA replication to begin. - These short strands of DNA serve as the starting point for DNA synthesis. - Forward and reverse primers - due to anti-parallelism. - DNA polymerase directed extension from primer binding sites. - rimers are designed to match segments of DNA we wish to copy. One primer will attached to the top strand at one end of the segment and the other primer to the bottom strand at the other end. - Ligation (joining) of all bases into a polymer.

Question 55

Quantitative PCR | Also known as real time PCR

Answer 55

- The initial quantity of DNA in the sample is detected by monitoring the growth phase of the reaction. - And measuring the cycle number at which the fluorescence intensity of the sample is determined. - Overcomes the background noise or threshold.

Question 56

PCR Denaturation

Answer 56

- When a DNA solution is heated sufficiently, the double- stranded DNA unwinds and the hydrogen bonds that hold the two strands together weaken and finally break. - This is referred to as the melting temperature or, the temperature at which one half of a particular DNA duplex will disassociate and become a single strand of DNA.

Question 57

Amplicons

Answer 57

Amplified DNA fragments which are the producs of the PCR.

Question 58

Annealing temperature Ta

Answer 58

This is the temperature, approximately 5oC below the lowest melting point of the base of primers used.

Question 59

Why are primers used?

Answer 59

- Primers are segments of DNA (or RNA) that are compliementary to a given DNA sequence. - Primers are needed in order to initiate replication by DNA polymerase.

Question 60

Reaction mix

Answer 60

* The more STR markers that are coded then the greater the amount of data in the resulting profile. * This allows DNA profiles taken now to be more discriminating which means it is rather unlikely that DNA samples would match advantageously. * To replicate and therefore amplify the STR markers sample is loaded into the thermal cycler with a reaction mix. * And then put through a series of heating and cooling steps which begins the copying process.

Question 61

What does the reaction mix consist of?

Answer 61

- A catalyst enzyme - Nucleotide bases (building blocks for DNA) - Fluorescebt probes/dyes. - These different coloured dyes correspond to various markers. The more colours equals more markers that can be used be used. - Generally speaking, four colours are used to mark against 17 markers as well as identifying whether the sample iss male or female.

Question 62

PCR Positive

Answer 62

- Is an application using known concentration input of DNA. - It is used to show a successful PCR reaction has occurred. - So, if any abnormalities seen any samples that were identified with that PCR – positive it is not due to inconsistency/incorrect PCR. - It can be used to monitor the performance of the thermocycler.

Question 63

Sequencing

Answer 63

* In order to see which allele pair is present at each marker the fragments, copied need to be separated. * This is done by size/molecular weight. The test is undertaken using capillary electrophoresis. * This works by creating an electrical field that the positively charged DNA will be attracted to. * The DNA moves away from the negatively charged cathode towards the positively charged anode. Smaller fragment the faster it will travel.A sensor identifies the strands as they go past the detection window which results in a series of different colour fluorescent peaks of different sizes.

Question 64

Final steps of DNA processing

Answer 64

* Computers are then used in order to translate the peaks into a series of numbered alleles (two for each marker). * In order to do this a control sample (referred to as an allelic ladder is run through the genetic analyser. * This is a sample of all available types of allele sequence for each marker.

Question 65

What is an allelic ladder?

Answer 65

* Allelic ladder is made up of DNA fragments that represent common alleles at a locus. * Off ladder (OL) alleles are those which size outside the categories represented in the ladder. * Internal size standards (ISS) are specific DNA fragments of known sizes which are defined and used to size unknown fragments.

Question 66

Locus

Answer 66

- A particular place on a chromosone or within the genone. - Refers to the place on the chromosome where the mutation (polymorphism) under consideration is located. - A locus may be defined as a single base pair at a particular point

Question 67

1. Amplified fragments of DNA. 2. Synonym for Real Time PCR. 3. Consisting of a catalyst enzyme, nucleotide bases & fluorescent probes. 4. The process of two strands of DNA rejoining. 5. A blank sample that is extracted using the same reagents and protocol, used to identify contamination. 6. Equipment used to filter, during DNA purification.

Answer 67

1. Amplified fragments of DNA are called amplicons 2. Synonym for Real Time PCR is quantitatice PCR. 3. A reaction mix consists of a catalyst enzyme, nucleotide bases & fluorescent probes. 4. The process of two strands of DNA rejoining is called annealing 5. A blank sample that is extracted using the same reagents and protocol, used to identify contamination is called extraction negative. 6. Equipment used to filter, during DNA purification is called a microcon.