DNA Analysis and Interpretation

Nucleobases general structure

Calculating a complementary sequence base pair pattern

Why is DNA a double helix?

Negatively charged phosphate groups repel each other

Base pairs hydrogen bond

Stacking of nucleobases through hydrophobic/Van der Waals interactions compacts duplex vertically

Calculating Duplex Stability

Buffers usage in DNA solution

• Ensures we have control over protonation state

• Prevents degradation

• Maintains a constant pH to control protonation, maintaining the consistent electrical charge of the molecules

• Maintains a constant temperature, preventing overheating and potential degradation of the DNA fragments

Henderson-Hasselbalch Equation

• To inverse a log, you do 10 to the minus of it superscript

Instrumentations to measure concentration of DNA

Gel Electrophoresis theory

• DNA is attracted to the positive electrode, due to the negative phosphate groups in the DNA backbone, attracting to the positive charge

• Movement is proportional to charge/mass ratio

• Samples are loaded, at the top

• Using a ‘ladder’ allows you to compare with known lengths

• Shorter strands move faster, appearing at bottom of gel

Visualising a gel

DNA absorbs UV light, so decreased transmission can be observed.

But UV light damages DNA rapidly.

Smeared bands, due to isotropic release of radiation.

32 Phosphorus labelling

Capillary electrophoresis

Used with many types of chemical and biochemical samples

Similar to HPLC, but force is electric field rather than pressure

For nucleic acids, capillary is filled with acrylamide gel

Obtain 1D graph, not 2D image

 

Faster than running a gel

Higher resolution than a gel

Can be automated

Impact and uses of PCR

Hereditary diseases

Identify viruses or microbes

Paleobiology

Familial relationships

Forensic investigation

PCR components

• Template

• Taq polymerase

Is a heat-resistant version of the polymerase enzyme

Stable for short periods of time at 90 degrees Celsius

Can replicate a 1000 base pair strand of DNA under 10s

 

• Primers

Polymerase needs primers as it can only add bases to pre-existing strands

Primer sequence needs careful design to ensure proper binding to the right side only, telling Taq polymerase where to begin, only 15-30 bases long

Primers made by solid phase phosphoramidite chemistry, giving us control to design and modify our primers to our needs.

Most of human DNA sequences same in every person

-          DNA profiling target repetitive, short tandem repeats (STRs, which are highly variable)

-          We design primers to target the edges of these STRs

-          Primer concentration determines the maximum yield of product; they are used up in each cycle.

• dNTPs

Building blocks of new DNA

Added to allow the Taq polymerase to ‘build’

Build off the 3’ end of the DNA

• Buffers and salt

Needed for hybridisation and extension

PCR process

Initialisation:

94 degrees Celsius for 30s – 5mins

Ensures template DNA is fully suspended and properly denatured

Especially important if the template is very long

Some ‘hot start’ polymerases require activation to begin working this way

Denaturation:

~ 94 degrees Celsius for 30s

Splits double stranded DNA into single stranded DNA

Annealing:

~ 64 degrees for 30s

Binds primers to template strands

Temp must be a few degrees lower than melting point of primers

Primers bind over complementary templates because of high concentration

Polymerase will also bind but not proceed

Elongation:

72 degrees for 30s

Or 1 min per 1000 base pairs

Synthesise the complementary strand

Temp is optimised for activity of the enzyme

Cycling:

15-40 repeats

Each cycle doubles DNA concentration

Too few = not enough amplification

Too many = limited by Dntp concentration

Too many leads to truncated products

Final extension and hold:

72 degrees for several minutes

Ensures all strands are finished

Reduces truncated products

Final hold – 4 degrees until needed, best condition for storing product

Troubleshooting - PCR

Occurs due to poor choice of primer sequence

Can occur if too much primer added

Primer dimer – when primer attaches to itself, and not to the DNA strand

Profiling vs Sequencing

Reading a Sanger Sequence

Visualising a Sanger Sequence

Use phosphorus-32 labelled ddnTPs, but hard work to read the sequence

So, instead of P-32, label each ddNTP with a different colour of fluorescent dye

Sanger Sequencing vs Next Generation Sequencing (NGS)

Why makes nucleic acids?

• New drugs

• Responsive healthcare

• Future manufacturing

Making nucleic acids + their uses

Synthetic cycle capping

Capping = killing unreacted strands

One coupling failure every 200 strands

Leads to deleted mutations

Primer may not bind

Error will be carried forward to protein synthesis

Other biochemistry altered

Example shown:

Synthetic cycle - protection + purification of oligonucleotides + PAGE purification

Synthetic cycle – protection:

Protecting non specific groups from attaching to the strand.

 

Purification of oligonucleotides:

Failed strands have to be removed

PAGE purification:

Cut out portion with strand ‘freeze and squeeze’

Visualising DNA - the EPG axis + interpretating peaks

• Graphic representation of DNA fragments

• Horizontal axis = time/size (base pairs)

• Vertical axis = intensity/quantity (Relative Fluorescence Units - RFU)

• Single peaks - homozygous, inherited same allele from both parents

• Double peaks - heterozygous, different alleles at the same locus

• Height of peak is proportional to the amount of DNA that gave rise to that particular peak during the PCR amplification process

How peak info is read

• Computers translate peaks into a series of numbered alleles, two for each marker

• A control sample, referred to as an allelic ladder, is run through the genetic analyser

• Analytical ladder is made up of DNA fragments that represent common alleles at a locus

• Off ladder alleles are those which size outside the categories represented in the ladder

• Internal size standards are specific DNA fragments of known sizes which are defined and used to size unknown fragments

Three main factors regarding DNA mixtures

• How many people contributed DNA to the mixture?

• How much DNA did each person contribute?

• How degraded is the DNA?

~ All these can increase the complexity of the mixture

Limit of Detection meaning

= the lowest amount of DNA at which a profiling system can reliably detect true genetic signal rather than background noise.

Environmental challenges - degradation + inhibition + ski slope

• Degradation = fragmentation of DNA due to heat, moisture, or UV

• Inhibition = contaminants (dyes, soils) that ‘block’ the PCR process

Different ways inhibitors can interfere with Taq polymerase:

• Binding to Taq polymerase, to its active site, preventing it from binding to DNA, dNTPs, carrying out strand extension

• Denaturing the enzyme, preventing it from binding

• Interfering with primer annealing, so weak peaks produced in the end

• High peaks on the left (small fragments), low/vanishing peaks on the right (large fragments), causes degradation or inhibition

• Both degradation and inhibition show a ski slope on the EPG

• Ski slope = technical artefact - gradual decline in baseline signal across the EPG

• Potential allele dropout at larger loci

Large and small molecular weights

• A big fragment of DNA, an allele that corresponds to a big amount of DNA is more likely to suffer from degradation than a smaller one in the same mix

  1. A big piece of DNA takes more time to go through the capillaries.

  2. It is harder to get amplified during PCR amplification process than a small one.

  3. If we’ve got something in the mix that is making it hard for the DNA polymerase is to amplify the DNA during PCR amplification that effect is going to be observed for the biggest pieces of DNA than it is for the smallest pieces of DNA

Comparison between SGM+ vs DNA 17 profiles

• Number of loci = 10 (+ amelogenin) vs 16 (+ amelogenin)

• Amelogenin = sex typing gene, whether a DNA sample came from a biological male or female

• Robustness against inhibitors

DNA 17 + why it is preferred over previous multiplex

• Enhanced DNA profiling system that examines 17 genetic markers (16 autosomal, 1 sex marker), replacing the older SGM+ system

• Only small amounts of DNA needed to begin with

• SGM+ loci = more sensitive

• Increased cycle number of PCR

• More resistant to inhibition

• Reducing adventitious random match probabilities

How the NDNAD finds matches - 3 possibilities

• Crime to person (direct hit)

• Crime to crime (linking scenes)

• Person to crime (backloading suspects)

Statistical foundations - maths behind the match

• Hardy Weinberg principles

• Formula 1 (homozygous) - p squared

• Formula 2 (heterozygous) - 2pq

Contamination + dealing with it + sources of it + opportunities for it to occur

• Contamination = to make something impure, by exposure to, or addition of, a polluting substance

• Wearing appropriate PPE

• Items brought into the lab wiped with alcohol

• All people who enter labs must provide elimination DNA samples, so if DNA matter found on substances then people can be disregarded if present in lab

• Each bench has its own equipment, reducing contamination between sets of items

• If undetected, contamination could affect the whole batch of samples in a gross or blanket contamination

• Sources: police, SOCO, pathologists, forensic scientists

• Opportunities: from person to stain from consumable to stain, from stain to stain

• Can occur at collection, extraction, amplification, injection. Degraded or low templated DNA increases risk

• Touch DNA prone to contamination

Adventitious transfer

• A ‘chance’ match where two people share a profile, highly rare

Analytical Threshold + stochastic threshold

• Analytical threshold = the RFU level where signal is distinguished from background noise

• Stochastic threshold = level where we can confidently say no ‘dropout’ has occurred

Any single allele above the ST would be considered homozygous, any allele below the ST is treated as having a missing sister allele

Deciphering nomenclature, e.g., D8S1179

• D = DNA

• 8 = chromosome 8

• S = Single copy sequence

• 1179 = registration number, variations in the repeat number

Allele dropout

• Describes when allelic peaks fall below the analytical threshold of an instrument.

• It is generally set to some amount above the lower detection limit of the instrument, where an analyst can reliably assign a peak as allelic with a low or nil risk of the peak being a baseline artifact.

• Low-level DNA template and/or degraded DNA results in alleles not amplifying above this threshold, resulting in incomplete or partial profiles

Mixture Interpretation and the Clayton Rules

~ When dealing with more than one contributor

• Identify the mixture

• Determine the number of contributors

• Estimate ratios and identify major vs minor contributors

Rules:

• All observed alleles accounted for

• No genotype introduces an allele that is not observed

• Use the minimum number of contributors needed to explain data

• Homozygotes must be justified by peak height

• Peak height ratios must be biologically plausible

• Dropout considered, but not over assumed

• Conditioning on known contributors must not distort mixture

The Laboratory Journey

• Firstly, cells harvested

Extraction:

• Breaking membranes (lysis), using a blender or pestle and mortar

• Key methods: Chelex, silica based

• Detergents added to dissolve proteins to free the DNA

• Goal is to maximise yield, while removing contaminants

Precipitation:

• Sodium ions added to neutralise the negatively charged DNA, then alcohol added to precipitate the DNA, forming a solid

Purification:

• DNA precipitate washed with alcohol to remove any impurities

• Centrifugation to separate debris, the pellet, from the DNA, supernatant

Quantitation:

• Determining concentration before the next step

• Amplification and detection

• Thermal Cycler - PCR machine

• Genetic analyser - the capillary electrophoresis equipment

• RFU - unit of measurement for peak height

Quality control protocols

• Ensures the results are valid

• Positive control - a known DNA sample to ensure PCR worked

• Extraction negative - blank sample processed alongside evidence, checking for lab contamination

Forensic History - RFLP era and DQ-Alpha and Blue Dots

• RFLP - restriction fragment length polymorphism

• Visual output: autoradiograms, resembling a barcode

• Much longer timescale - weeks, compared to days now

Early PCR methods:

• DQ-Alpha: used test strips

• Blue Dots: the specific visual marker

Quantitative Analysis - peak height ratios (PHR)

• Checking for balance

• Calculation: smaller peak/larger peak x 100

• Standard: must be above ~ 60-70% to be ‘in balance’

• Imbalance causes: mixture of two people, or stochastic effects (low DNA)

Forensic Workflow Overview

• Evidence recovery and extraction

• Quantification and Amplification

• Capillary Electrophoresis (CE)

• Data Analysis (EPG)

Artefacts meaning + types

= things that happen during testing that cannot be reproduced, are not reflective of the DNA that is associated with a sample

Biological artefacts - stutters:

• Small peak positioned just before a large clear peak, signifying where a larger peak would appear

• Occurs due to the DNA polymerase moving slightly forward and backward, causing a a PCR product which is slightly shorter than true fragment

• Stutter filters used to remove stutters on the software

~ Stutter, imbalance, drop-in and dropout are all stochastic effects

Instrumental artefacts:

Spikes and blobs:

• Peak on an EPG that is too tall and narrow relative to what we would expect to see for a peak in the range of all the other peaks

• If peak is short and squat, it is an indication of a blob

Phadebas testing

• Detection of saliva stains

Real time vs standard PCR

1. Real-time PCR aka quantitative PCR (qPCR) allows for the monitoring of amplification of a targeted DNA molecule during the PCR process.

2. 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.

3. 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.

4. 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.