DNA Variation and Degradation

Question 1

Topics to be covered

Answer 1

• Types and causes of genetic variation
• Types and causes of DNA degradation
• How can we take advantage of genetic variation to aid forensic investigations

Question 2

State the reasons which contribute to making us unique to one another

Answer 2

Sexual reproduction
Recombination
Mutation

Question 3

How does recombination make us unique?

Answer 3

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.

Question 4

Name some factors which lead to mutation in DNA and contribute to our uniqueness

Answer 4

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.

Question 5

Do identical twins have identical DNA?

Answer 5

No
Due to different mutation events
e.g., Methylation events are environmentally cause so they have an effect

Question 6

What can mutations be due to?

Answer 6

Spontaneous or enzyme catalysed changed

Question 7

How different are we from one another?

Answer 7

• 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

Question 8

State the different types of genetic variation

Answer 8

Length polymorphisms
Structural variation
Epigenetics
SNPs

Question 9

Tell me about length polymorphisms and an example

Answer 9

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

Question 10

Tell me about structural variation and what this includes

Answer 10

Structural variants including** copy number variation **
o Different copies of sections of DNA
o Segments of at least 1000 bases

Question 11

Give some examples of epigenetic changes

Answer 11

Epigenetics
o E.g., methylation (adding methyl groups to DNA)
o E.g., Histone changes

Question 12

What are SNPs?

Answer 12

o Single nucleotide polymorphisms
o Single base change at a position which is different in individuals

Question 13

What was the 1000 genomes project?

Answer 13

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

Question 14

With Length polymorphism/Tandem repeats, what is estimated and what does it determine?

Answer 14

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)

Question 15

Tell me about Short tandem repeats (STRs) and what it determines?

Answer 15

• 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

Question 16

How does variation occur in these STR?

Answer 16

DNA polymerase slippage

Question 17

Explain DNA polymerase slippage

Answer 17

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

Question 18

Whats the mutation rate with STRs, where it occurs and some general information regarding it?

Answer 18

• 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

Question 19

Tell me about DNA17 and its key markers to note

Answer 19

• 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

Question 20

Tell me about the controversy thats been seen in the field between linked markersand its use in forensics

Answer 20

• 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.,)

Question 21

With structural variants, compared to the reference genome, an average genome has how many…
- deletions
- inversions
- copy-number variants
- what else…

Answer 21

o Roughly 1000 large deletions
o Roughly 10 inversions (flipped DNA)
o Roughly 160 copy-number variants
o + other types e.g., Alu insertions

Question 22

Tell me about copy number variation and its use in forensics

Answer 22

• A DNA segment of at least 1kb
• Variation in copy number within a population
• **Not used in forensics **

Question 23

All cells have the same DNA sequence, but a skin cell is different to a pancreatic cell, why?

Answer 23

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)

Question 24

Does epigenetic mutations change the DNA sequence?

Answer 24

No

Question 25

What other research is ongoing regarding epigenetics and its use in forensics?

Answer 25

* 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.

Question 26

What are SNPs?

Answer 26

* Single nucleotide polymorphisms * Nucleotide: building blocks of DNA * Polymorphism: Variation * Single base variation between individuals at a particular point in the genome

Question 27

What are most types of SNPs? Tell me the different types of conversions and the most common type?

Answer 27

* 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

Question 28

Tell me about a type of C-->T transition

Answer 28

**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

Question 29

In order for an SNP to be considered a true SNP what % of the population does it have to be present in?

Answer 29

* 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%

Question 30

What regions can SNPs be present in?

Answer 30

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

Question 31

What has a higher mutation rate, SNPs or STRs?

Answer 31

STRs

Question 32

Why do SNPs have 2 alleles and not 4?

Answer 32

 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

Question 33

DNA degrades when exposed to what?

Answer 33

**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

Question 34

What does DNA degradation occue as a result of and tell me about each

Answer 34

* 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

Question 35

What type of degradation occurs with apoptosis?

Answer 35

Enzymatic and non-enzymatic

Question 36

Tell me about enzymatic degradation

Answer 36

* 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

Question 37

Tell me about the types of non-enzymatic degradation

Answer 37

* **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 ## Footnote 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

Question 38

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?

Answer 38

STRs routinely used in forensic casework can require amplification of nearly** 45bp**

Question 39

DNA degradation can lead to what and what are some strategies to target this?

Answer 39

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)

Question 40

What is there a greater chance of success with when coming to generate a DNA profile with degraded DNA, SNPs or STRs?

Answer 40

* 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

Question 41

Example of human traits with DNA variation

Answer 41

* 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

Question 42

Where can DNA variation be used in forensics?

Answer 42

* **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 **

Question 43

**Conclusions**

Answer 43

* 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