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Generation of DNA profiles Flashcards

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1
Q
A
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2
Q
  1. Thermo-stable polymerase
  2. The unzipping enzyme in DNA replication
  3. This strand is synthesized in fragments
  4. Used to initiate DNA synthesis
  5. DNA fragments sealed together by this enzyme
  6. Will add to free DNA nucleotides
A
  1. Thermo-stable polymerase os taq polymerase.
  2. The unzipping enzyme in DNA replication is helicase.
  3. The lagging strand is synthesized in fragments.
  4. Primers are used to initiate DNA synthesis .
  5. DNA fragments are sealed together by the ligase enzyme.
  6. DNA polymerasr will add to free DNA nucleotides.
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3
Q

RFLP

Restriction fragment length polymorphisms

A
  • It was the first DNA used for DNA.
  • It was good for side-by-side comparisons.
  • It used an xray plate to mark where the DNA bands were through radiation.
  • These tests were used back in the 1980s and 1990s.
  • They took about a month sometimes two or three months to generate a profile.
  • Very discriminating.
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4
Q

DQ alpha

A
  • The first PCR-based tests were called HLA DQ - Alpha
  • Sometimes referred to as poly-marker
  • In these tests the critical information comes from a series of strips which were called test strips.
  • In these tests what scientist were looking for was the presence or absence of these blue dots.
  • The particular pattern of dots would give us some insight in terms of what sorts of DNA molecules are present in a particular sample.
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5
Q

Improvements of DQ alpha compared to RFLP.

A
  • The first generation of DNA test took blood stain about the size of 50p piece to obtain a result.
  • They would often take months to produce – whereas the second -generation tests could work with about 100th of that amount
  • DQ Alpha could work with bloodstains were almost too small to be seen by the naked eye
  • The results could be generated much more quickly – (half a day).
  • Quicker and requires less sample.
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6
Q

DQ alpha versus RFLP

A
  • DQ Alpha was more sensitive, in terms of the amount of starting material that was necessary in order to generate a profile.
  • RFLP were not overly good in terms of sensitivity.
  • Neither were RFLP overly good in terms of the speed at which profiles could be obtained, they were nevertheless very discriminating.
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7
Q

Multi-Locus Probe

MLP

A

In 1985 Sir Alec Jeffrey’s developed a form of DNA profiling called Multi-Locus Probe (1985) or MLP.
Whilst MLP was a significant development the method was:
* Lengthy
* Required a large amount of sample to produce a profile
* Results were complex to interpret

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8
Q

Single Locus Probes

SLP

A
  • By 1987 a process called Single Locus Probes or SLP (VNTR) was in routine.
    The results were:
  • Easier to interpret compared to the previous MLP method.
  • Initial results could be obtained within a few days although it often took over a week to prepare a full profile
  • However, this method still lacked sensitivity
  • Gave poor results with degraded samples
  • It was also difficult to resolve mixtures containing DNA for more than one person.
  • This is when we moved away from gel technology and we moved towards repeating number sequences.
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9
Q

What did RFLP allow?

A

RFLP tests did enable scientists to assign matching probabilities in the order of one in 1 million or one in 10 million as the chance of a match.

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10
Q

Match probability of DQ alpha

A
  • Statistics would typically be described in terms of one in a few hundred or maybe one in a few thousand.
  • DQ Alpha were much better in terms of sensitivity and the time needed to generate a result, but they didn’t deliver as much in terms of discriminating power as the previous RFLP system.
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11
Q

Sensitivity definition

A

The amount of starting material or the size of the stain we can begin working with.

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12
Q

Discrimination power

A

Power to distinguish between one thing and another.

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13
Q

What is the criteria for judging the benefits of a DNA profiling system?

A
  • Discriminating power
  • Sensitivity
  • Ability to deal with artefacts
  • Speed
  • Ability to deal with mixtures
  • Ability to conduct database searches
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14
Q

Artefacts

A

Artefacts are things that could have got into the DNA profile and contaminated it.

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15
Q

Mitochondrial DNA

A
  • Mitochondrial DNA - more sensitive than the automated STR’s.
  • The main reason for this is that whilst every cell within it contains two copies of all of our genetic material; one copy we get from our mother and one from our father
  • In each mitochondrial will have multiple copies of what is referred to as mitochondrial DNA.
  • Mitochondrial DNA is relatively small, relative to the amount of DNA that is present in the nucleus and so not as much information contained within.
  • Nevertheless, it is present in very high copy numbers and so gives us the chance to get a result from even a tiny fraction of a cell.
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16
Q

Mitochondrial DNA drawbacks

A
  • The other drawback with mitochondrial DNA is that all mitochondrial is maternally inherited.
  • In simple terms, what this means, is that an individual inherits all of their mitochondria from their mother and her mother in turn obtained all her mitochondria from her mother etc.
  • The test are very sensitive but not as discriminating because we know where we find one kind of mitochondrial DNA there will be many more maternal relatives who will share this genetic material.
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17
Q

Y STR

A

-The focus of these tests is on the Y STR markers which reside on the human Y chromosome.
- Females have two X chromosomes whereas males have an X and Y.
- There is information on the Y chromosome that can be analysed by forensic scientists particularly where we think there is a possibility of a male contributor to an evidence sample.
- It might give us some insight into what that males DNA profile look like without being distracted by things which may have been contributed by a female .
- Helpful feature when we are talking about a mixed sample, for example those with a sexual offence – when we have a large amount of female DNA mixed with a small amount of a suspect.
- There are parallels between Y STR testing and mitochondrial DNA testing. These are, – where is mitochondria are maternally inherited – Y chromosomes are exclusively paternally inherited.

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18
Q

How much DNA do we need?

A
  • The kind of DNA that we leave behind in a fingerprint is roughly 100 cells and is plenty to generate a DNA profile using the widely available commercial DNA assays
  • Therefore, we do not need a lot of material to start, and we can obtain a DNA from almost everything that has come into contact with an individual.
  • Each cell contains between 6 and 7 pg. of DNA.
  • DNA profiling kits generally recommend using between 500 and 1,000 picograms of template DNA which roughly works out to around 100 to 200 cells.
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19
Q

How much DNA do we need now?

A
  • The new DNA 17 multiplex is can generate profiles with much less starting material
  • The DNA 17 test requires about 80 cells (around 500 pg.) DNA for a standard result
  • DNA profiles have been obtained from as few as 15 cells when these are in good condition.
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20
Q

Number of cells transferred from wearing a garment?

A

The number of cells which might be transferred by the action of wearing a garment is well over 1,000. At 6 pg. per cell that this will provide more than 6,000 pg. of DNA, easily enough to obtain a full DNA profile.

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21
Q
  1. DNA that is maternally inherited?
  2. The threshold of detection with DNA 17 is in the region of?
  3. 1 millionth of a litre?
  4. Quantity of DNA left behind in a finger mark?
  5. Synonymous with DQ alpha?
  6. Paternally inherited DNA sequences can be found here?
A
  1. DNA that is maternally inherited is mitochondtial.
  2. The threshold of detection with DNA 17 is in the region of 500pg.
  3. 1 millionth of a litre is a microlitre.
  4. Quantity of DNA left behind in a finger mark is arounf 100 cells.
  5. Synonymous with DQ alpha is polymarkers.
  6. Paternally inherited DNA sequences can be found in Y-STR.
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22
Q

Polymorphism

A
  • Regions od DNA which greatly differ from person to person.
  • The non-coding parts of the DNA.
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23
Q

Why does DNA differ from person to person?

A
  • Simply because the DNA in that region is likely to come in many different forms and sizes.
  • There are many areas of the DNA genome which are the same between individuals, for example those which code for eyes and hair and limbs etc.
  • These areas are not going to be any use for distinguishing between people and so we would need to focus on the polymorphic areas which are likely to be different.
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24
Q

What do we mean by locus/loci?

A
  • Loci reside on specific parts of the chromosome.
  • It is a specifc spot we pay attention to on the length of a persons DNA sequence.
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25
Q

What are markers?

A
  • Markers are the points we’re looking at that have variation.
  • They have different known frequencies at different locations.
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26
Q

Allele

A
  • Allele is a term used to describe a specific region of DNA which varies between people.
  • At each locus we will see two alleles (assuming heterozygous).
  • One alleles inherited from each parent.
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27
Q

Discrimination power in terms of alleles

A
  • What’s the chance of the allele being at that location in relation to the channel population.
  • The discrimination powe is the ability to determine the probability of an allele being present at that location.
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28
Q

PCR in depth

A
  • We target and amplify the STR markers in the sample.
  • The process of PCR requires the generation of two specific primer sequences that are complementary to a sequence of DNA upstream and downstream of the STR marker of interest (these are often called “forward” and “reverse” primers). Attached to one of the primers is a fluorescent dye.
  • The FAM fluorescent dye is one of the most commonly used dyes and will emit a colour signal in the blue portion of the visible spectrum.
  • With each cycle of PCR, the targeted amplicons (the products of PCR) are copied through a process in which the double-stranded DNA is opened,
  • The primers bind to their targeted sequences,
  • An enzyme (Taq polymerase) then extends the newly formed sister strand.
  • The resulting product is two copies of DNA from the one initial amplicon. This is repeated over and over, and with each cycle, the amount of targeted DNA within the sample is doubled (an exponential amplification). At the end of the PCR process, millions of amplicons are generated, all having the blue FAM dye attached.
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29
Q

First step in automated STR testing?

A
  • When we are doing DNA testing the objective is to get to DNA – genetic material from a reference sample and compare this to a crime scene sample.
  • The first step in this process is to extract and purify the DNA from both of these two different types of material (the reference sample and the crime sample).
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30
Q

What is the purpose of DNA extraction?

A
  • This is to obtain a sample solution of DNA which has no other impurities and inhibitors. - The extraction nowadays is carried out using the Qiagen kit.
31
Q

How is DNA extraction done?

A
  • Cell lysis releases the DNA into solution by denaturing the cell walls and breaks down the peptide bonds and to avoid enzyme degradation with the addition of ATL and proteinase K.
  • Then, the DNA is being isolated – this removes the cell debris by adding 100% ethanol which also helps the DNA bind to the silica column.
  • After that, the DNA is purified to remove the impurities and inhibitors.
  • Elution buffer is added to break the H bonds formed between the silica column and the DNA.
32
Q

Filtering DNA

A
  • Once the cell has been lysed and the enzymes have broken down different proteins, DNA molecules need to be fully isolated.
  • This is done through adding different reagents and ethanol to the solution and then filtering the solution.
  • As different chemicals have been used to isolate the DNA, it is vital that DNA purification is conducted to ensure that there are no impurities or inhibitors in the DNA sample.
33
Q

Purification of DNA

A
  • At this stage different reagents which were added to filter CPG is removed by cutting out the supernatant.
  • Ammonium hydroxide is removed by evaporation and protecting groups are removed by desalting the column or ethanol precipitation.
  • It is also important, in this stage to remove any free strands; these can be cut off using the target strand.
34
Q

Quantification of DNA after purifcation

Intercalating dyes

A
  • Once purified, the DNA must then be quantified to establish the concentration of the DNA. This can be done by adding fluorescent dye to the sample.
  • This dye tends to be flat, positively charged molecule which is complimentary to the phosphatase in the DNA structure.
  • Due to the slow the process of interpolation, the dye inserts itself between the nucleotide bases in the DNA strand.
  • Adding these intercalating dyes/probes also allows for PCR progression to be observed.
  • Intercalating dye - sometimes referred to as fluorescent probes which insert themselves between the successive bases in DNA.
35
Q
  1. Specific region of DNA which varies between individuals?
  2. Gene loci are found at specific areas on?
  3. Amplified fragments of DNA?
  4. A commonly used primer dye
  5. Enzyme which extends the newly formed system strand during PCR?
  6. Breaks the hydrogen bonds between silica column and DNA.
A
  1. Specific region of DNA which varies between individuals are alleles.
  2. Gene loci are found at specific areas on chromosomes
  3. Amplified fragments of DNA are amplicons.
  4. A commonly used primer dye is fluorescein amidite.
  5. Enzyme which extends the newly formed system strand during PCR is taq polymerase.
  6. The elution buffer breaks the hydrogen bonds between silica column and DNA.
36
Q

Why is PCR important?

A
  • Because it provides a means for copying and increasing the amount of DNA available.
  • It means that a DNA profile can be obtained from very small amounts of starting material.
37
Q

What happens after each copy is made in PCR?

A

As each new copy is made, it is labelled with a dye so that it can be visualised in the laboratory.

38
Q

What was the first DNA profiling method using PCR called?

A

QUAD

39
Q

dNTPs function

A
  • Essentially form the building blocks for the new DNA strands.
  • There are four different types: DATP, DCTP, DGTP, DTTP.
  • The extension step, DNA polymerase adds free dNTPs from the reaction mixture to the template DNA.
40
Q

Buffer for polymerases

A
  • Most commercial polymerases come supplied with their ideal buffer.
  • These buffers not only supply the correct pH, but they always have additives like magnesium, potassium, or DMSO, which help optimize DNA denaturing, renaturing, and polymerase activity.
41
Q

Taq polymerase

A
  • Taq polymerase also binds to the template strand at the start of the area for amplification but doesn’t proceed until the temperature is increased. The temperature in the apparatus is increased to 74°C for around 30 seconds.
  • This is the optimum temperature for the enzyme, Taq polymerase and is known as the extension phase where the DNA polymerase synthesises new complementary DNA strand by extending the primers along the whole amplification area.
  • Hydrogen bonds formed between the nucleotide bases of the DNA template strand and a new complementary strand, forming the DNA double helix.
42
Q

Annealing and extension

A
  • The stages of denaturation and annealing and extension repeat over and again for roughly between 15 and 40 repeats.
  • This is to ensure that there is sufficient amount of amplification of the DNA. Once the specified amount of repeat have been satisfied, the DNA sample must undergo a final extension which occurs at 72°C for circa seven minutes.
  • This is important to ensure that all strands have been fully amplified and finished as well as to remove any truncated products from the sample.
  • The final stage is called the final hold in which the DNA sample is held at 4°C.
43
Q

Capillary electrophoresis

A
  • This is done once the DNA has been amplified.
  • This apparatus contains capillary tube which contains a matrix.
  • The capillary tube is a narrow tube where the capillary electrophoresis occurs.
  • There is a detector placed close to the positive side of the apparatus.
  • This will record when DNA fragment passes the detector and the time it passes. The detector is on the side of the apparatus as the DNA is negatively charged and so when electric field is applied to sample the fragments will migrate towards the positive reservoir. The larger fragments will move slower through the capillary matrix on small fragments and other recorded on the EPG.
44
Q

How does capiallry electrophoresis start?

A
  • As DNA is a negatively charged molecule, we can start off the DNA process at one end of the capillary by injecting the material into the tube.
  • By applying an electric field, it will naturally be drawn to the positive electrode due to the electric current.
45
Q

How does the DNA move through the tube in capillary electrophoresis?

A

In capillary electrophoresis DNA is pulled through the tube using an electric current from negative electrode to positive electrode.

46
Q

What happens to bigger molecules in capillary electrophoresis?

A
  • Big molecules of DNA find it more difficult to travel through the capillary tube and the matrix within; medium-sized molecules will move more easily and the ones which move through quickest of all will be the smallest fragments
  • Eventually and regardless of their size – small medium or large they are all going to pass by the detector window within the genetic analyser.
47
Q

What is the detector windown in capillary electrophoresis?

A

It is wihtin the genetic analyser and is a camera paired with a laser.

48
Q

What happens to the DNA fragments as they move past the laser in capillary electrophoresis?

A

As DNA fragments move past the laser they become excited and fluoresce with different colours because of the labelling added in the PCR application and those colours get picked up by the camera.

49
Q

What happens once the DNA moves past the detector window in capillary electrophoresis?

A
  • Eventually all the DNA will move past the detector window and this information is then transmitted to a computer.
  • The raw data resembles something like a line graph with various peaks moving from left to right.
  • This is where the information is for our DNA profile.
50
Q

The raw data in electrophoresis

A
  • The vertical axis identifies the relative fluorescence units or RFU’s.
  • These are measures of the intensity of light picked up by the camera in the detector window at different points in time.
  • The horizontal axis is measured in terms of minutes or seconds.
  • It starts at time zero and proceeds left to right all the way through to around 30 minutes from beginning to end.
51
Q

The raw data in electrophoresis

First peaks

A
  • For around the first 10 or 12 minutes or so you might see little reaction we then see different lines of different colours which the camera has detected.
  • The first peaks represent the primers which are left over from the PCR step in the process and after they have moved through then we start to see this pattern of peaks – representing our DNA profile.
52
Q

Capillary electrophoresis

The electrophereogram

EPG

A
  • This information is captured by a computer and then computer software is used to pull apart the peaks into their component colours.
  • The software is also used to attach names to each of these different fragments or peaks in each of these graphs.
  • These electropherogram’s, from the raw data produced from the genetic analyser
53
Q

When is allele droput typically seen in an EPG?

A
  • The threshold of detection may cause allele dropout or small peaks.
  • This is seen towards the end of the profile.
  • These are the bigger bits which are more susceptible to degradation.
54
Q

Why do some peaks appear small in EPG?

A

Some peaks are small due to artefact

55
Q

Why are the relatively balanced peaks in EPG?

A

This is because we inherit the same volume of DNA from both our parents.

56
Q

Homozygous peak

A
  • Often taller because you get the same amount of DNA from mum and dad in the same peak.
  • Occurs when the same allele appears twice.
57
Q

Reading an EPG

A
  • The peaks that we see on the electropherogram each correspond to individual alleles.
  • In some tests there are around nine different loci with peaks which indicate which alleles are present at this locus.
  • We will have some peaks in blue, some peaks in green some in yellow (often shown in black for contrast purposes). There are some peaks shown in red and so we have four different coloured fluorescent dyes to label the DNA in the PCR application process.
58
Q

How are EPGs read?

A
  • Within each of the three first colours – namely the blue green and the yellow we will see that there are clusters of peaks, three blue three green and three yellow. These will of course increase with the later techniques using more loci but for the time being letters use the SGM plus (10 marker system) for ease of understanding.
  • ## These correspond to the fragments of DNA which have been amplified from specific loci that are part of this particular test and each of those loci has a name.
59
Q

Non polymorphic amiogenin gene

A

If we have a DNA profile with two peaks at the amilogenin locus this would illustrate that this comes from an individual with both an X and a Y chromosome – in other words a male DNA profile.

60
Q

One peak at the amilogenin locus

A

ONe peak (just x) indicates a females DNA.

61
Q

Internal size standards in EPG

A
  • These are not defined during the PCR step but are already pre-added to the multiplex and are internal size standards by which we can compare with peaks in the yellow green and blue lines above in order to make size determinations.
62
Q

Known standards in EPG

A

Known standards in EPG allow us to position other alleles

63
Q
  1. In cell lysis, the DNA is released into solution after denaturing the cell walls and breaking down?
  2. Instrument used in centrifugal filtration?
  3. After centrifugation what name do we give to the clear liquid line above the solid residue?
  4. Term used to explain how the dye inserts itself between the nucleotide bases in the DNA strand?
  5. An enzyme that synthesizes long chains of polymers a nucleic acids?
A
  1. In cell lysis, the DNA is released into solution after denaturing the cell walls and breaking down peptide bonds.
  2. Microcon is the instrument used in centrifugal filtration.
  3. After centrifugation the clear liquid line above the solid residue is called the supernatant.
  4. Interpolation is the term used to explain how the dye inserts itself between the nucleotide bases in the DNA strand.
  5. Polymerase synthesizes long chains of polymers a nucleic acids?
64
Q

Diploid

A

We have two copies of our genetic instructions.

65
Q

Homozygous

A

Sometimes we get the same instructions from both a mother and father and that would make is homozygous at a particular locus.

66
Q

Heteroygous

A

Sometimes we get different alleles from mother and father and that would make is heterozygous at a particular locus.

67
Q

What else can we determine from the EPG?

A
  • The position of the peaks also tells us something. Peaks on the left side of the EPG represents the small fragments of DNA and the ones that are on the right side represents the largest fragments of DNA.
  • The larger alleles will always appear on the right.
  • Sometimes a peak may appear homozygous but one of the alleles has dropped out so its actually heterozygous.
68
Q

Peak height in EPG

A

The height of the peak is proportional to the amount of DNA that gave rise to that particular peak during the PCR amplification process.

69
Q

Alternative hypothesis

Coincidental match

A
  • The chances of this happening with a good, clean STR results are very small and, as we report in the UK, one in 1 billion.
  • So, the chances of coincidental match can, in many instances it pretty much effectively ruled out.
70
Q

Hypothesis regarding DNA results

Evidential weighting

A

Hypothesis = the reason the DNA profile matches that of the individual is because they have the same source.

71
Q

Alternative hypothesis

Error in handling

A
  • Maybe there is been some sort of error in the process maybe there was a mistake in the collection of the evidence maybe an error in the handling after the collection.
  • Maybe there’s been a mistake in the manipulations within the laboratory such that there is been some type of contamination.
  • Maybe there was a mistake or an accident or deliberate mistakes which are all possibilities which must be considered when we are deciding upon the weight of evidence to be applied in a particular case.
72
Q
  1. During the process of purification where are the impurities to be found after centrifugation?
  2. What is the process which occurs between extraction and PCR?
  3. In the process at KFS what step is undertaken using ATL and Proteinase K?
  4. The process of PCR is undertaken in the?
  5. The process of electrophoresis is undertaken in this equipment?
  6. On the EPG, the height of the peaks (Y axis) is measured in?
A
  1. During the process of purification the impurities are found in the pellet after centrifugation.
  2. Quantification occurs between extraction and PCR.
  3. In the process at KFS cell lysis is undertaken using ATL and Proteinase K.
  4. The process of PCR is undertaken in the thermal cycler.
  5. The process of electrophoresis is undertaken in a genetic analyser.
  6. On the EPG, the height of the peaks (Y axis) is measured in RFU.
73
Q
  1. RNA structure is?
  2. RNA shares some bases except thymine which is swapped with?
  3. DNA is found in nucleus &?
  4. RNA forms in the nucleus and then moves to?
  5. Holds and replicates genetic instructions
  6. Reads & translates DNA to a format that can be used to build proteins?
A
  1. RNA structure is single stranded.
  2. RNA shares some bases except thymine which is swapped with uracil.
  3. DNA is found in nucleus & mitochondria.
  4. RNA forms in the nucleus and then moves to the cytoplasm.
  5. DNA holds and replicates genetic instructions.
  6. RNA reads & translates DNA to a format that can be used to build proteins.

DNA Analysis & Interpretation (19 decks)

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