DNA Profiling Flashcards
LO
Have an in-depth knowledge of:
o The history of DNA profiling
o Short tandem repeats (STRs)- their classification and purpose
o The steps involved in generating a DNA profile
Name two people who are key to the background to DNA profiling
Sir Alec Jeffreys
Colin Pitchfork
How did Sir Alec Jeffreys contribute to DNA profiling?
o Pioneer of DNA fingerprinting
o 10th September 1984- Has a ‘eureka’ moment in his lab in Leicester when looking at an X-ray film image of DNA from different members of his technician family
o Restriction enzymes to analyse DNA: restriction fragment length polymorphisms (RFLPs)
o This showed both similarities and differences between the individuals
How did Colin pitchfork contribute to DNA profiling?
o First person convicted of murder using DNA fingerprinting
o 2 counts of sexual assault and murder, one in 1983 and the other 1986,victims: Lynda Mann and Dawn Ashworth
o Caught because of mass screening
o Arrested: 19th September 1987
o Sentenced to life imprisonment: 22nd Jan 1988
DNA fingerprinting
Crime scene comparison
What is a tandem repeat?
Tandem repeats occur in DNA when a pattern of one or more nucleotides is repeated and the repetitions are directly adjacent to each other.
It is estimated that how much of the human genome is made up from repetivitve elements of DNA?
50%
What does the type ofsequence being repeated determine?
The classification given to the stretch of repetitive DNA
What is satellite DNA and what are the types of it?
Satellite DNA consists of very large arrays of tandemly repeating, non-coding DNA. Satellite DNA is the main component of functional centromeres, and form the main structural constituent of heterochromatin.
types: microsatellite (STRs), minisatellite (VNTRs) and alpha satellite DNA. These constitute the tandem repeats
What is alpha satellite DNA?
Alpha satellite DNA –> 171bp unit
o Combination of higher-order repeat units (HORs: repeated in tandem) and stretched of disorganised monomers
o Found primarily at centromeres of chromosomes
o Up to 10% of the human genome
Tell me about minisatellite DNA
Minisatellite –> **10-100bp unit **
o Variable number tandem repeats (VNTRs)
o E.g., D1S8 (MS32) –> 29bp (12-800+ repeats)
Up to 2.3x104 bp
o The larger the target site the harder it is to amplify!
o Analysed using digestions- large amounts of DNA required
Tell me about microsatellite DNA
- Arrays of 5-10 repeats
o 20-200 bp
o Short tandem repeats (STRs)
What makes up the Variable number of Tandem repeats (VNTRs)?
The micro and minisatellite DNA
With Microsatellites…
How many bp in each repeat?
What type of repeats?
- 2-10 bp in each repeat
- Di, tri, tetra, pentanucleotide repeats
Short tandem repeats, what are they?
STRs are short repeated sequences of DNA of about 2-6bp
They account for 3% of the human genome
The number of repeat units is highly variable amongst individuals, which offers a high power of discrimination when it comes to analysis for identification purposes
What causes Tandem repeat variation? and what type does this occur in?
Misalignment at meiosis
Satellite and mini satellite
**Reading frame mistakes at meiosis **
Micro-satellite
How does variation occur in STRs?
DNA polymerase slippage
* Low complexity DNA sequences like microsatellites (mono,di, tri repeats) and minisatellites are associated with mutagenesis in both eukaryotic and prokaryotic genomes
* These types of sequences are characterised by high instability, consisting of the addition/ deletion of repeated units, leading to variation in repeat copy number
* Such genetic variations have been termed “dynamic mutations”
* Arrest of the replication machinery within a repeated region is associated with such instability,where primer and template become misaligned.
* this process is known as replication slippage, is involved int he generaton of deletions/ insertions within repeat regions
https://www.frontiersin.org/articles/10.3389/fmicb.2014.00403/full
What are some types of STRs and how they can be denoted
Simple –> identical length and sequence
TH01 (AATG)n
Compound –> Two or more adjacent simple repeats
D2S1338 (TGCC)n(TTCC)n
Complex
D21S11(TCTA)n(TCTG)n(TCTA)3TA(TCTA)3TA(TCTA)2TCCATAn
Incomplete/ non-consensus repeat units –> simple or complex STRs
TH01 9.3 allele: (AATG)6ATG(AATG)3
9 tetranucleotides and one incomplete trinucleotide repeat at the 7th repeat site= 9 total repeats but one with 3 therefore 9.3
STR nomenclature answer the following…
Where was it found?
How is it written systemically?
STR profiles: genotypes
Visualisation of alleles…
What is meant by heterozygous and homozygous?
Heterozygous: 2 different versions of the same gene
Homozygous: 2 versions of the same gene
Can different people have different combinations of alleles (genotypes)?
Yes
Importance of STR allele frequency
What are the stages to the processing of DNA evidence?
Highlight the stages which are requires to generate a DNA profile
What are the key stages to the generation of a DNA profile?
What is important to ensure for each step?
* DNA extraction
o DNA must be broken out of cells and separated from all the other cellular material to be compatible with downstream processes
o Any inhibitors must also be removed
*** DNA quantification **
o Amount of amplifiable DNA must be quantified to ensure optimal PCR
* DNA amplification **
o Target amplification of forensically relevant markers(e.g., STR loci)
**
* Amplified product separation and detection
o Identification of the individuals’ alleles to generate a profile for comparison
What is the history of PCR?
* Kary Mullis
o 1983: First described PCR method
o 1985: First PCR paper published in Science
Very labour intensive because the first type of polymerase used in the reaction had to be replaced at every step
o 1988: Mullis’ second PCR paper published in Science
First use of thermostable polymerase, Thermes aquaticus (Taq)
o Kary Mullis wins the 1993 Nobel Prize for chemistry
Define PCR
**PCR: the polymerase chain reaction **
“An enzymatic process in which a specific region of DNA is replicated repeatedly to yield many copies of a particular sequence)
PCR is exponential growth (double products)
What are some properties of DNA that are useful to know when it comes to PCR?
o Unlike proteins, **DNA can be denatured, re-natured, then denatured… **
o Able to be heated to extremely high temperatures, breaking the H bonds between bases (denature), then cooled allowing the H bonds to reform
o The temperature at which DNA is half-denatured is called the Melting temperature, TM (related to the CG content of it and is related to the number of H bonds present between the pairs)
How many bonds between each pair of complementary bases?
What effect would increasing/ decreasing the number of H bonds have on the melting temperature?
** How many bonds between each pair of complementary bases? *
A-T= 2
C-G= 3
** What effect would increasing/ decreasing the number of H bonds have on the melting temperature? *
Increasing the number of H bonds makes the melting temperature higher as more energy is required to break the bonds
What are the components of a PCR reaction and what are they used for?
* DNA primers
o Short oligonucleotides flanking the region to be copied (STR)
o Complementary to the DNA sequence
**
* DNA polymerase enzyme**
o Uses all components to replicated DNA; must be thermostable as using lots of temperature changes to denature DNA and don’t want to denature our enzyme else it won’t work again
*** Magnesium chloride (MgCl2) **
o Enzyme co-factor
o Needs the optimal amount to function
*** Buffer (Salts: KCl and Tris-HCl) **
o Control pH environment
*** Deoxynucleotide triphosphates (dNTPs) **
o Building blocks
o Nucleotides wit triphosphate added and can be added into the new chain
*** Template DNA **
o Where do we get this? can we add our sample direction to the reactions? How much should we add?
Need to get DNA out of the cells
Haem is an inhibitor of PCR
Need to make sure DNA is free from cells and all inhibitors are removed from reactions
Kinetic relationships help determine the quantity of DNA needed
o Need to know the DNA we are copying
*** De-ionised water **
o Why is regular water not good enough?
The presence of H has ions
So, ions don’t interfere with enzyme function
Potential contamination in tap water
Need sterilised and de-ionised water
What is thermal cycling and what are the steps which occur in a cyclical fashion?
What happens in each stage of PCR?
**1. denaturing stage **
* The template DNA and the other core ingredients are heated to 95˚c
* These temperatures cause the Hydrogen bonds to break between the complimentary bases
* The double helix separates
* This leads to the production of two single strands of DNA
* It is important that the temperature is maintained at this stage to ensure that the DNA has been seperated completelt
2. annealing stage
* During this stage the reaction is cooled to 45-65˚c
* This enables the primers to attach to a specific location on the single-stranded template DNA via Hydrogen bonding
* The exact temperature depends on the Melting temperature of the primers you are using (depending on CG content)
* The primers are single strands of DNA or RNA that are 20-30bases in lengh
* The primers are designed to be complimentary in sequence to short section of DNA on each end of the sequence to be copied they bind in the forward and reverse direction
3. extending stage
* During this final step, the heat is increased to 72˚c to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases in the 5’-3’ direction
* The temperature for strand elongation will vary depenidng on the specific function of th enzyme
Tell me the type of amplification of DNA from PCR
Exponential amplification
* Every cycle generates roughly 2n molecules of DNA (n=cycle number; assuming 100% efficiency)
* PCR can be used to amplify a target product
* Real-time/ qPCR: uses exponential amplification to quantify how much template DNA there is based on DNA standards of the known concentration
What are important considerations when working with PCR products?
- Important to never cross-contaminate
- Amplified products are virtually impossible to get rid of
- Must keep all components of the lab separate (preferable physical separation):
o Pipettes, tips, reagents, gloves, waste, equipment, bench space etc.
o When working with low-level DNA, this includes clothing, footwear, books, paper, pens: some labs require wearing hair nets, facemasks, use of disposable scalpels and tweezers
What temperature is DNA denatured at?
Therefore what is needed during PCR reactions?
- DNA is denatured in PCR at temperatures of **95˚c **
- Polymerase is a protein. What happens to produce at high temperatures?
- Need a polymerase enzyme able to withstand high temperatures
- Original PCRs required more enzyme added at each cycle to prevent inactivity
Name and tell me about a thermostable polymerase
- In 1976 polymerase was isolated from bacteria Thermus aquaticus (Taq) – nothing done about it for years
- Bacteria found growing in hot springs of Yellowstone national park
- 10 years later, PCR developed- Mullis released he needed a thermostable polymerase to withstand high temperatures and remembers discovery
What temperature is polymerases optimal activity at?
What is the problem with this?
How can this be overcome? Name two methods
Polymerases optimal activity at roughly 72˚c (Note elongation temperature of PCR cycling conditions)
**Problem: **exhibit activity below this temperature creating potential problems with reactions
**Solutions: **
o Development of AMPLITaq Gold (ABI)- enzyme is chemically altered to make it inactive at room temperature. Needs to be incubated at high temperatures to become active (Note incubation step cycling conditions)
o Use hot start PCR: Taq is added to PCR once temperature of reaction has gone above roughly 60˚c. Why?
Asides for time consuming, what other problems could there be?
* Contamination from opening tubes to add Taq
Tell me the PCR cycling conditions and stages?
Name and describe some controls for PCR
* Positive control: method to check reaction effectiveness or quality of the reaction components
* Negative control: check for contamination of any of the reaction components
* Extraction negative: check for contamination in any of the extraction components
- Good laboratory practice should include at least a positive and negative control run with each set of reactions
What are the two ways that DNA synthesis can occur and tell me what is required in each ?
What are the advantages of PCR?
- Start with very small amounts of DNA
- Use a wide array of sample types
o Simple extraction procedures - Target specific DNA sequences of interest (less than 2000 bases)
- Fast and robust
- Commercial kits available
What are the disadvantages of PCR?
- Sensitivity to contamination and inhibitors
- Non-specific amplification possible
- Mutations in primer binding sites (no amplification)
- Stochastic effects from low levels of template
- Primer design can be complicated, particularly with multiplexes (primer-dimer, etc)
- PCR optimisation required
History of PCR in forensics
** 1990:** Forensic PCR identification methods start
*** 1992-1995: **FBI starts casework with PCR for 3 markers and Amelogenin
o OJ Simpson trial makes public aware of DNA evidence, PCR and mullis
* 1996-1999: First multiplex PCR STR kits developed and validated
* 2000: several labs stop RFLP testing in favour of STRs and PCR based methods
STR amplification and primer design
Note: tetranucleotide repeat
What are some things to consider when selecting an STR?
**1. Is it polymorphic? Different versions of alleles **
2. Is it suitable for amplification with PCR? **
a. Number of bases in repeat –> tetranucleotide is best
b. Size range –> not too large
c. Primers –> unique and consensus sequence (every person has this consensus sequence for primers to attach, same for everyone)
**
3. Is it robust?
a. Invariant primer binding site
b. Reliably amplified
c. Slippage
Polymerase slippage in PCR
- Also happens in PCR reactions
- 2>3>4>5
- Dinucleotide repeats not used in forensic science: process of polymerase slippage can also occur during PCR. This is more common in smaller repeating unit
- Trinucleotide repeats now dropped for use in forensic science: still an error rate
- Tetra and Penta repeats favoured
What are Pseudoautosomal regions?
The pseudoautosomal regions, PAR1, PAR2, are homologous sequences of nucleotides on the X and Y chromosomes
Outside of the PAR some genes are shared,tell me about one
Amelogenin:
o Codes for a protein found in tooth enamel
o The X-chromosome version (AMELX) has a 6bp deletion in intron 1
Amelogenin detection
History of STR systems
-
1994 forensic science service:
o “quad” the first multiplex 4 STRs - 1995- “SGM’ (second generation multiplex) 6 STRs
- **1999- **SGM plus 10 STRs
-
2001- USA CODIS multiplex 13 STRs
o Combined DNA index system -
2009/10-Next generation multiplexes (NGM) released
o Expanded to 12 STRs (European standard set) - 2014- DNA17 introduced in England and Wales
DNA17 tell me about it
- DNA17 is a method that analysis 16 DNA STR loci
- This is 6 more than previous kits which increases the power to match or eliminate a suspect greater than every before
NB: a Loci is a location within a genome (such as a gene or another DNA segment of interest)
How do we detect and differentiate between our PCR products- especially if we have 16 STR markers?
o Use capillary electrophoresis (CE) to separate DNA amplicons based on size
o Multiple fluorescent dyes used to detect amplicons (incorporated into PCR primers)
o Increase in number of markers targeted means systems need to be able to detect up to 6 dyes
DNA17 uses 5 dyes
Amplicon structure
Some things to consider with STR kits
* Marker selection
o Polymorphic markers
o No linkage or linkage disequilibrium between markers
*** Primer design **
o Should be in a non-variable region (same in all individuals)
o Chosen so that all alleles are separable by size (and dye colour)
o No interactions between primers (such a primer-dimers)
* Method development:
o Optimal annealing temperature for all primers
o Optimal number of cycles (can be decided in-house)
Multiplexing using size and dyes
Name and tell me about an STR kit
STR profile- DNA17
What is the purpose of the Staff elimination database (SED) ?
In case the staff contaminate the sample, you are able to eliminate them
Why have we moved to analysing STRs instead of DNA fingerprinting?
Issue with DNA: time consuming and needed more DNA to start with, relief on digestion rather than amplification which isn’t best for forensic
Why must we not use the same equipment pre- and post- amplification?
contamination
Why is it important to calculate STR allele frequencies?
For statistical evaluation
Why must we use data from unrelated individuals?
Little variation in those related as they’ll share alleles
What are some key features that we need to consider when choosing an STR to analyse?
o Polymorphic
o Consensus
o Robustly amplify
How many STR loci are targeted in DNA17 analysis?
16 + 1 amelogenin to make DNA17
