DNA Variation and Degradation Flashcards
Topics to be covered
- Types and causes of genetic variation
- Types and causes of DNA degradation
- How can we take advantage of genetic variation to aid forensic investigations
State the reasons which contribute to making us unique to one another
Sexual reproduction
Recombination
Mutation
How does recombination make us unique?
Recombinant DNA is the general name for a piece of DNA that has been created by combining at least two fragments from two different sources.
Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, and differ only in the nucleotide sequence within that identical overall structure. Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species, like the mythical chimera.
R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends.
Name some factors which lead to mutation in DNA and contribute to our uniqueness
Epigenetics e.g., methylation
Toxins
Oxidative damage (oxygen free radicals produced from ATP production which are very reactive towards DNA and cause it to change)
UV damage
etc.
Do identical twins have identical DNA?
No
Due to different mutation events
e.g., Methylation events are environmentally cause so they have an effect
What can mutations be due to?
Spontaneous or enzyme catalysed changed
How different are we from one another?
- Any two unrelated humans of the same sex will show about 99.9% concordance in their DNA sequences
- 0.1% difference between individuals which is key interest in forensics
State the different types of genetic variation
Length polymorphisms
Structural variation
Epigenetics
SNPs
Tell me about length polymorphisms and an example
Length polymorphisms (e.g., STRs), length variation in the DNA
o Tend to occur in areas of repetitive DNA
o Our genome has about 50% of our genome being repetitive DNA
o Some variation found due to length polymorphisms
Tell me about structural variation and what this includes
Structural variants including** copy number variation **
o Different copies of sections of DNA
o Segments of at least 1000 bases
Give some examples of epigenetic changes
Epigenetics
o E.g., methylation (adding methyl groups to DNA)
o E.g., Histone changes
What are SNPs?
o Single nucleotide polymorphisms
o Single base change at a position which is different in individuals
What was the 1000 genomes project?
1000 genomes project (set up to sample 1000 genomes of people in population)
* Managed to samples 2504 individuals from 26 worldwide populations
* Found over 88 million variants characterised
o 84.7 million SNPs
o 3.6 million indels (insertions or deletions)
o 50,000 structural variants
* Typical individual differs from the reference human genome at 4.1-5 million sites
* Over 99% are SNPs or short indels
With Length polymorphism/Tandem repeats, what is estimated and what does it determine?
Length polymorphisms- Tandem repeats (tandem repeats= adjacent repeats)
* It is estimated that nearly 50% of the human genome is made up from repetitive elements of DNA
* The type of sequence being repeated determines the classification given to the stretch of repetitive DNA (e.g., alpha satellite, minisatellites, microsatellites)
Tell me about Short tandem repeats (STRs) and what it determines?
- Size of repeat **determines length polymorphism classification **
- In forensics mainly use STRs/microsatellites
- 2-10 base pairs in the repeat (try to use 4bp repeats in forensics)
- Arrays of 5-50 repeats
o 20-200 bp
How does variation occur in these STR?
DNA polymerase slippage
Explain DNA polymerase slippage
o Polymerase falls off DNA
o DNA falls apart and reattaches with polymerase
o But sometimes repeat in template or new strand is looped out
o This means if original was 7 repeats, then the new is only 6 repeats
o Mutations as changes number of repeats (STR mutation)
o Sometimes rectified by repair enzymes but sometimes not
Whats the mutation rate with STRs, where it occurs and some general information regarding it?
- The average mutation rate for STRs is often quoted as roughly** 2-3 mutations for every 1000 STR loci passed on **
o This **high mutation rate **means that between different people there is high variation which is good for individualisation in forensics - Recently more specific data shows that rates can vary considerably between STR loci and even between alleles
- Vast majority of all these mutations involve the loss or gain of 1 repeat unit (97%)
- Harder to look for women than men as for men you can use Y chromosome data, and the age of the father also has an impact
Tell me about DNA17 and its key markers to note
- Used in England and Wales
- 16 STR + sex determining marker on X and Y chromosome
- Scotland uses a multiplex of >20
- 2 markers on 2 but opposite end so any recombination occurs then they are likely to be separated
-
STR within non-coding region of gene (chromosome 4)
* SE33 (chromosome 6) is very complex and it’s good to separate people, has long alleles - Chromosome 12: vWA and D12S391 which is a short marker, easy to amplify and quite varied, however both markers are close on chromosome which is not ideal
Tell me about the controversy thats been seen in the field between linked markersand its use in forensics
- e.g., B and C would have similar markers to those generations past due to similar location and proximity
- Caused problem in forensic as two markers which are close together in DNA17 (vWA and D12S391)
**D12S391 and vWA controversy **
* 12 million bases apart on population level won’t make too much of a difference (difference between vWA and D12S391)
* Enough chance of recombination that they will be fine
* Those markers 50 million base pairs apart are considered different enough
(paper in FSI genetics by Phillips et al.,)
With structural variants, compared to the reference genome, an average genome has how many…
- deletions
- inversions
- copy-number variants
- what else…
o Roughly 1000 large deletions
o Roughly 10 inversions (flipped DNA)
o Roughly 160 copy-number variants
o + other types e.g., Alu insertions
Tell me about copy number variation and its use in forensics
- A DNA segment of at least 1kb
- Variation in copy number within a population
- **Not used in forensics **
All cells have the same DNA sequence, but a skin cell is different to a pancreatic cell, why?
o Part due to methylation **
o Due to promotor region (before gene), CpG site**, if C methylated (CH3 group on it), then it won’t start the process. Most genes are methylated as don’t want to produce all protein possible in each gene
o In tissue can have genes which are not fully turned on/off
o Sometimes methylation can be used in crime scenes to help identify body fluids i.e., the proteins generally found in that said body fluid
Of particular importance in sexual assault cases and to help determine what has occurred in that crime scene
Amanda Knox trial (watch)
Does epigenetic mutations change the DNA sequence?
No
What other research is ongoing regarding epigenetics and its use in forensics?
- Research ongoing regarding uses such as determining which tissue DNA came from, ageing etc. (another use with methylation)
- Age of person which left blood stain with an error of 3 years
All to do with different genes which are turned on/off as you age i.e., energy, cancer etc.
What are SNPs?
- Single nucleotide polymorphisms
- Nucleotide: building blocks of DNA
- Polymorphism: Variation
- Single base variation between individuals at a particular point in the genome
What are most types of SNPs?
Tell me the different types of conversions and the most common type?
- Most SNPs are C/T SNPs or** A/G SNPs **
- Easier to swap A–>G than C–>T
- Transitions are more common than transversions even though there are more combinations of transversions which could occur
Tell me about a type of C–>T transition
Cytosine deamination
* One of the ways these transitions/ transversions occur
* Deamination occurs by adding water: which removes ammonia (one H from water added to NH2 to enable its removal)then the remaining OH of water add onto nucleotide
* Hydrolytic deamination of cytosine to uracil generates a highly mutagenic DNA base lesion and is considered one of the major sources of spontaneous mutation in living organisms.
* 5-methylcytosine–> thymine via deamination also
* Sometime nucleotide is methylated which in the case of 5-methylcytosine produces a DNA nucleotide base
Read more into
In order for an SNP to be considered a true SNP what % of the population does it have to be present in?
- To be considered a true SNP, the minor allele must exist in at least 1% of people
- HapMap estimate there are around 10 million SNPs with a worldwide minimum allele frequency of >1%
What regions can SNPs be present in?
Coding and non-coding regions SNPs
o Most in non-coding regions
o Those in coding regions can have an effect of phenotype or disease predisposition of an individual
o Forensics generally looks at non-coding
What has a higher mutation rate, SNPs or STRs?
STRs
Why do SNPs have 2 alleles and not 4?
Due to rarity of base mutating
Genetic drift
Less useful than STRs for identification as only have 2 alleles and major/minor so less variation
Need to look at >50 SNPs to get same identification then with STR
DNA degrades when exposed to what?
**Heat or the elements **
- In 1988, Bar et al., (FSI 39(1):59-70) shows that DNA would degrade if exposed to fire or the elements for a suitable time
- More recently (Alaeddini et al., FSI: genetics, 2010), a review of DNA degradation has examined the mechanisms by which this happens
What does DNA degradation occue as a result of and tell me about each
- Degradation occurs due to cell death which occurs either through **apoptosis or necrosis **
o Apoptosis is controlled and if cells have energy, they will choose to kill cells. Various method of degradation in apoptosis
o Necrosis occurs when don’t want it to, occurs when no energy in cell to do anything
What type of degradation occurs with apoptosis?
Enzymatic and non-enzymatic
Tell me about enzymatic degradation
- Removal of histone proteins will enable endonucleases (digest DNA from within) to cleave DNA
- Endogenous nucleases or exogenous nucleases (e.g., from bacteria)
- More than 70% of soil microorganisms contain nucleases
Tell me about the types of non-enzymatic degradation
- Hydrolytic reactions: attack of water, attack specific bonds which are most susceptible such as the sugar base bond
- DNA crosslinking: when DNA will form link to other DNA or proteins
- **Oxidative reactions: **ROS
- **Radiation: **damage DNA
If too many breaks or short chains it won’t work when trying toreplicate on PCR as there is fewer bp so unable to get full profile
Degradation of DNA results in short DNA fragments rather than long moleucles, STRs routinely used in forensic caseworkcan require amplification of nearly how many bp?
STRs routinely used in forensic casework can require amplification of nearly** 45bp**
DNA degradation can lead to what and what are some strategies to target this?
Results in **locus dropout or amplification failure **
Various strategies for dealing with degraded DNA. These include (aim to reduce amplicon size)
o MiniSTRs (useful with disaster victim identification, terrorism attacks etc.). aims to shorten amplicons. Redesign primers and move closer to repeating region then may help to amplify markers in less bases
o **MtDNA **(mitochondrial DNA is less susceptible to degradation)
o SNPs (only 1bp of interest, so could design primers of say 20 bases each, meaning only would need 40bp of DNA bases)
What is there a greater chance of success with when coming to generate a DNA profile with degraded DNA, SNPs or STRs?
- In highly degraded samples there is a greater chance of obtaining an intact fragments 100bp long to act as a template than of 300bp for an STR
- Should be far greater success rate with SNPs than STRs on degraded samples for this reason
Example of human traits with DNA variation
- Meta-analysis of the heritability of human traits based on fifty years of twin studies
- Results provide compelling evidence that all human traits are heritable: not one trait had a weighted heritability estimate of 0
- Trying to use in forensics to see if DNA can predict the phenotype of an individual in cases with no witness or witness which may have been killed
Where can DNA variation be used in forensics?
- Molecular autopsy: especially in young people into why they have died
- Genetic genealogy: look at millions of SNPs, compare to databases which are voluntarily on internet, to work out if someone did a murder is related to that in who have done ancestry test. Ethics and legalities involved
- **Diseases: **identify if someone has a particular disease. Won’t do due to practicalities and ethical reasons
- **Environmental factor (epigenetics–> lifestyle factors): **i.e., metals. Look at bones from bodies and in different areas of world contributes to different isotopes in a person from drinking water/ food to help identify where they came from. E.g., cigarettes or other lifestyle factors
- Biogeographic ancestry: use SNPs. Look at where ancestors came from before recent population movement happened
- Phenotyping: pigmentation, hair colour, eye colour, height, facial prediction etc. (my group)
- Genetic modification
- **Psychological profile **
- **Wildlife forensics **
- **Domestic wildlife forensics: **cats and dogs’ hair in crime scene from suspects it came from. Meow plex to identify cat hair.
- Meta-genomics: don’t just look at human DNA within sample, also DNA, viruses, plant etc. which can help identify location
- **Epigenetics and age prediction **
Conclusions
- We can harness DNA variation in forensic science for many applications
- One factor affecting which type of variation we use (the markers we choose), is the quality of the DNA
- With more novel applications, care must be taken when using these powerful tools, especially with respect to ethics and result interpretations
