Appendix 1 Forensic parameters

19 Apr

Appendix 1   Forensic parameters

Match probability

The probability that the two randomly selected individuals will have identical genotype.

where,pM isthematchprobability,pk representsthefrequencyofeachdistinctgenotype, m is the number of distinctive genotypes. The combined probability of match over several loci is the product of the value for all the loci.

Power of discrimination
The probability that two randomly selected individuals will have different genotypes.

where, pD is the power of discrimination of a single locus, pM is the match probability of a single locus, PDcomb is the power of discrimination of several loci, PDi is the individual locus power of discrimination. The “  ” sign stands for multiplication.

Power of exclusion
The fraction of the individuals that is different from that of a randomly selected indi-vidual. It can also be defined as the power of a locus to exclude a person being the biological father. Thus the value differs in each case. The average for a locus is the power for a single locus.


where, h is the heterozygosity and H is the homozygosity at the locus.
For several loci the formula is:

where L is the number of the loci, PEl is the exclusion probability for the lth locus and
the   sign stands for multiplication.

Polymorphic information content
Indicates the polymorphic level of a locus.

where, pi is the frequency of each distinct allele, and n is the number of distinct alleles.

Appendix 2   Useful web links

Professional bodies and agencies

ANZFSS – The Australian and New Zealand Forensic Science Society, Inc.
http:// ENSFI – European Network of Forensic Science Institutes. FBI – Federal Bureau of Investigation: FBI Laboratory. lab/labhome.htm FSS – The Forensic Science Service (UK). ISFG – International Society for Forensic Genetics: INTERPOL – The International Criminal Police Organization.
http://www.interpol. int/ NIFS – National Institute of Forensic Science Australia. home.html The Forensic Science Society (UK).

Statistical analysis
Forensic  Mathematics:  Contains  information  of  the  kinship  software  with
DNA·ViewTM and articles/discussions focused on the statistical/mathematical in-
terpretation of DNA profiles.

GDA: a statistical software package that computes linkage and Hardy-Weinberg
disequilibrium, some genetic distances, and provides method-of-moments es-
timators for hierarchical F-statistics.

Powerstats: A Microsoft Office Excel-based tool for calculating descriptive
statistics  and  forensic  parameters  for  STR  loci.

ENFSI DNA WG STR Population Database: calculates the profile frequency of a
SGM Plus profile using 24 European allele frequency databases.

Genetic markers and population databases

STRBase – Short Tandem Repeat DNA Internet DataBase: contains a large amount of information on STR polymorphisms. YHRD – Y Chromosome Haplotype Reference Database: a searchable database of Y chromosome STR haplotypes. EMPOP – Mitochondrial DNA Control Region Database: a collection of searchable mtDNA control region haplotypes from all over the world. MITOMAP -A compendium of polymorphisms and mutations of the human mito- chondrial DNA. ALFRED – The ALlele FREquency Database: contains allele frequency data for a wide range of genetic polymorphisms in different populations.

Commercial providers
All these companies provide a wide range of products in addition to the ones noted. Applied Biosystems: suppliers of kits and equipment for the analysis of STR and SNP polymorphisms. Promega Corporation: suppliers of kits for STR analysis and a wide variety of molec- ular biology products. Qiagen: suppliers of widely used DNA isolation kits. Whatman® : suppliers of FTA® card.

Allele: alternative forms of a gene or section of DNA at a given genetic locus. Allelic Drop Out: non detection of an allele at a given locus. This results in only one of the two alleles being detected at a heterozygous locus. Allelic Ladder: a mixture of all the common alleles at a given locus. The allelic ladder allows comparison with the unknown alleles and assists in allelic designation. Amplifiable fragment length polymorphisms (AMP-FLPs): polymorphic loci where alleles differ in the number of tandem core repeats. Alleles are typically between 500 and 1,000 bp in length. An example of an AMP-FLP is the locus D1S80. Autosome: a non-sex chromosome. In humans there are the 22 pairs of autosomal chromosomes; these do not include the X and Y sex chromosomes. bp: base pair – two complementary nucleotides in double stranded DNA. Adenine pairs with thymine and guanine pairs with cytosine. Chromosome: a single molecule of double stranded DNA associated with proteins to form highly ordered structure. Chromosomes are located in the cell nucleus of eukaryotes and are visible with light microscopy only during cell division when they become highly condensed. CODIS loci: the FBI defines a set of 13 STR markers for use in forensic analysis. Diploid:presenceoftwochromosomalsetsinacellandthereforecontainingtwocopies of the genome. DNA polymerase: an enzyme that catalyses the formation of a complimentary DNA strand in the 5′ to 3′ direction acting on a template DNA strand. Electrophoresis: separation of charged molecules through a matrix. DNA is negatively charged and will migrate from the cathode (-ve) to the anode (+ve) when an electric current is applied across the matrix. Euchromatin: part of the chromosome that is loosely packed in the interphase of the cell cycle. Most of the transcribed regions of the genome are located within the euchromatin.

Gene: a functional part of DNA that encodes a protein or RNA molecule. Gene Frequency: the relative frequency (proportion) of a gene in a population. Genome: the entire haploid complement of DNA in a cell or organism. The human genome comprises approximately 3.2 billion bases. Genotype: the particular set of alleles present in each cell. At any one locus the two alleles define the genotype such that if there are only 2 alleles then there are 3 possible genotypes (AA, AB, BB). Haploid: presence of one set of chromosomes in a cell and therefore containing one copy of the genome. Hardy-Weinberg Law (HW law): a law stating that in an ideal population, the frequen- cies of an allele will remain constant from one generation to the next. Heterochromatin: a highly condensed part of the chromosome which remains tightly packed throughout the cell cycle and is predominantly non-coding. Heterozygous: having different alleles at any particular locus. Homozygous: presence of two identical alleles at a given locus. Locus: the physical position of a gene or section of DNA on a chromosome. The plural of locus is loci. Locus Drop Out: the non detection of both alleles at a given locus. Low Copy Number PCR: a more sensitive method of DNA profiling where the number of cycles is increased in order to amplify amounts of DNA typically less than 100 picograms. Mitochondria: small semi-autonomous organelles of the cell which are primarily re- sponsible for energy production. They have their own circular genome that is 16 569 bp in length. Mutation: alteration in the DNA sequence. The most common form of a mutation is a single base transition (see below). Oligonucleotide: short sequence of single stranded DNA. Phenotype: physical form of an organism resulting from genetic traits and environ- mental factors. Polymerase Chain Reaction (PCR): a process of enzymatic amplification of DNA in vitro. Posterior Odds: in terms of a court case these are the odds of guilt of a defendant after the presentation of all evidence. It is the posterior odds that the judge or jury consider when coming to a verdict.

Primer: oligonuclceotide that binds to complimentary single stranded DNA and acts as a priming site for the initiation for the synthesis of the complementary strand by DNA polymerase. Prior Odds: in terms of a court case these are the odds of guilt of a defendant before the presentation of forensic evidence and may be considered as based on the non-scientific evidence. Purine: nitrogenous bases found in nucleic acids. The purine bases are adenine and guanine. Pyrimidine:nitrogenousbasesfoundinnucleicacids.Thepyrimidinebasesarecytosine and thymine. Restriction enzyme: enzyme with endonucleic activity that cuts DNA at specific se- quences. An example is Eco RI that cuts within the sequence GAATTC. Short tandem repeat (STR): polymorphic region of DNA where alleles differ in the number of tandemly arranged core repeats. STR alleles typically range in size between 100 bp and 400 bp. Also known as microsatellites. Single Nucleotide Polymorphism (SNP): the occurrence of two (or more) alleles at a single base position within the genome. SNPs are typically biallelic: transitions (see be- low) are more common than transversions (see below). To be considered as a population level SNP polymorphism the rare allele should be at a frequency greater than 1%. Southern blot: a technique that is used to transfer DNA from a gel onto a nylon membrane. Telomere: the terminal regions at the tips of the chromosomes. Satellite DNA forms much of the telomeric regions. Theta (θ) Statistic: a measurement of inbreeding within a population. Theta is often used interchangeably with FST in forensic genetics. Transition: the change in a base from a purine (see above) to a purine (A/G) or a pyrimidine (see above) to a pyrimidine (C/T). Transversion: the change in a base from a purine to a pyrimidine (A or G to C or T) or a pyrimidine to a purine (C or T to A or G). Variable number tandem repeat (VNTR): polymorphic region of DNA where alleles differ in the number of tandemly arranged core repeats. VNTR alleles can range in size from around 500 bp to over 20 kb. Also known as minisatellites.

A: Adenine – A purine base. One of the four bases of DNA
AMP-FLPs: Amplified fragment length polymorphisms
bp: Base pair
C: Cytosine – a pyrimidine base. One of the four bases of DNA
CODIS: Combined DNA Index System
DNA: Deoxyribonucleic acid.
EDTA: Ethylenediaminetetraacetic acid
FBI: Federal Bureau of Investigation of the USA
FSS: Forensic Science Service of the UK
G: Guanine – a purine base. One of the four bases of DNA
kb: Kilobase, a string of a thousand DNA bases.
LINE: Long interspersed elements
LTR: Long terminal repeat
MLP: Multi-locus probe
PCR: Polymerase chain reaction
RNA: Ribonucleic acid.
SINE: Short interspersed elements
SLP: Single locus probe
SNP: Single nucleotide polymorphism
STR: Short terminal repeat
T: Thymine – a pyrimidine. One of the four bases found in DNA.
VNTR: Variable number tandem repeat

Units of measurement

α-amylase 22
ABI PRISM ®  genetic analyzers 55, 116
ABO blood grouping system 2
absorption-inhibition ABO typing 2
adventitious hits 99
agarose gels 33-4, 35
ceiling principle 79-80, 83
drop-out 68, 69, 73
specific hybridization 119
allele frequency databases 78-83
allele ceiling principle 79-80, 83
Balding size bias correction 80, 81, 83
profile ceiling principle 82, 83
selection 83
subpopulations 80-2, 83
allelic ladders 58-61
amelogenin locus 53
3-aminophthalhydrazide 19-20
AMP-FLPs see amplified fragment length
AmpF/STR ®  see Identifiler ® ; PowerPlex ®
system; SGM Plus ®
amplified fragment length polymorphisms
(AMP-FLPs) 39
AmpliTaq Gold ®  40-1, 44
Balding size bias correction 80, 81, 83
Bayesian approach
evaluation process 92-3, 95
kinship testing 109-11
biological samples 17-25
collection 3, 19, 20-1
contamination 19, 20
identification and characterization 19-20
presumptive testing 21-3
reference samples 23
sexual and physical assault 21
sources 17-19
storage 23-4
biological samples 3, 17-20, 21, 23-4
DNA quantification 32
grouping 2, 121
polymerase chain reaction 44, 46
bones 31-2, 72-3, 129
buccal swabs 23, 46
Cambridge Reference Sequence (CRS) 129
capillary electrophoresis (CE) 54-5, 57-8, 118
CE see capillary electrophoresis
Chelex ®  100 resin 27-9, 31, 34, 45
chi square tests 78
chromosomal organization 7-9
CODIS see Combined DNA Index System
cold cases 101-2
Combined DNA Index System (CODIS) 52,
biological samples 19, 20
cold cases 102
polymerase chain reaction 45-8
Crick, F. 7
CRS see Cambridge Reference Sequence
D-loop 126
DAPI 33, 35
ddNTPs see dideoxynucleotide triphosphates
defendant’s fallacy 94
degraded DNA 71-3, 101-2
deionized formamide 55
deoxynucleotide triphosphates (dNTPs) 8, 42,
deoxyribonucleic acid (DNA)
extraction 27-32
challenging samples 30-2
Chelex ®  100 resin 27-9, 31, 34, 45
FTA ®  paper 29-30
general principles 27-30
hair shafts 31
hard tissues 31-2
phenol-chloroform-based 29, 31
seminal fluid 30
silica based methods 28-9, 31, 36
quantification 32-6
agarose gels 33-4, 35
deoxyribonucleic acid (DNA) (Contd.)
fluorescence spectrophotometry 35
hybridization 35
real-time polymerase chain reaction 35-6
ultraviolet spectrophotometry 34
replication 40
structure 7-15
chromosomal organization 7-9
coding and regulatory sequence 9-10
extragenic DNA 10
forensic genetics 11
genetic diversity 11
human genome 9-10, 11
short tandem repeats 13, 14
single nucleotide polymorphisms 13-14
tandem repeats 10, 12-13, 14
transposons 10
deoxyribose 8
4′,6-diamidino-2-phenylindole 33, 35
dideoxynucleotide triphosphates (ddNTPs)
dinucleotide repeats 66
dithiothreitol (DTT) 30
DNA see deoxyribonucleic acid
DNA IQTM Isolation System 36
dNTPs see deoxynucleotide triphosphates
DTT see dithiothreitol
EDNAP see European DNA Profiling Group
epithelial cells 17-19, 22, 33
ethidium bromide 33, 35
European DNA Profiling Group (EDNAP) 103
evaluation process 87-95
Bayesian approach 92-3, 95
defendant’s fallacy 94
frequentist approach 88-9, 94-5
hierarchies of propositions 87-93
likelihood ratios 89-92, 95
prosecutor’s fallacy 91, 93-5
exact tests 78
extragenic DNA 10
familial searching 100-1
flanking DNA 12
labelling 54
spectrophotometry 35
formamide 55
frequentist approach 88-9, 94-5
FTA ®  paper 23-4, 29-30
GeneMapperTM ID 56, 59, 67
GeneScan ®  56, 58, 67
genetic diversity 11
Genotyper ®  59-60
ancestry 120
clustering 132
follicles 17-19, 23
shafts 31
haplotype frequencies 130-1, 132
Hardy-Weinberg equilibrium (HWE)
allele frequency databases 78-83
deviations 76-8
infinitely large populations 76-7
migration effects 77
mutations 77
natural selection 77
random mating 77
statistical tests 77-8
heteroplasmy 127-9
heterozygosity 138
hierarchies of propositions 87-93
Bayesian approach 92-3, 95
frequentist approach 88-9, 94-5
likelihood ratios 89-92, 95
homoplasmy 127-9
homozygosity 78, 138
genome 9-10, 11
karyotype 9
remains 1, 111-12
lineage markers 129
short tandem repeats 71-3
HWE see Hardy-Weinberg equilibrium
hybridization-based methods 35
hypervariable regions 126-7, 129
Identifiler ®                               105,120,52,61
inbreeding 81-2
infinitely large populations 76-7
inhibition of PCR 44-5
internal-lane size standards 56-8
Interpol Standard Set of Loci (ISSOL) 103
interspersed repeats 10
ISSOL see Interpol Standard Set of Loci
Jefferson, Thomas 131
Jeffreys, Alec 2-3
Kastle-Meyer (KM) 21
kinship testing 1, 105-14
absorption-inhibition ABO typing 2
Bayesian approach 109-11
complex relationships 111
human remains 111-12
likelihood ratios 105, 109-10
lineage markers 131
paternity testing 105-11
Punnett squares 106-7
subpopulations 111
KM see Kastle-Meyer
Landsteiner, Karl 2
Lattes, Leone 2
LCN see low copy number
leuco-malachite green (LMG) 21
likelihood ratios
evaluation process 89-92, 95
kinship testing 105, 109-10
short tandem repeats 78
statements 91
lineage markers 125-35
applications 127-30, 131-2
copy number 125
hypervariable regions 126-7, 129
inheritance 125
interpretation 129-30, 132
mitochondrial genome 125-30
polymorphisms 126, 130-2
Y chromosome 125, 130-2
LINEs see long interspersed elements
LMG see leuco-malachite green
local Southern method 57-8
locus drop-out 68, 69
long interspersed elements (LINEs) 10
long terminal repeats (LTRs) 10
low copy number (LCN) PCR
contamination 44
degraded DNA 101-2
short tandem repeats 68-70, 73
LTRs see long terminal repeats
luminol 19-20
match probability 137
statements 88-9
migration effects 77
mitochondrial genome 111, 117, 125-30
mixed profiles 70-1
molecular autopsy 121
Mullis, Kary 3-4
multi-locus exact tests 78
multiplex development 51-3
muscle tissue 72-3
mutations 77
national DNA databases 97-104
adventitious hits 99
cross-border 103
United Kingdom 97-102
United States 102-3
natural selection 77
non-template-dependence 66-7
off-ladder peaks 58-9, 60
overloaded profiles 68
PACE see Police and Criminal Evidence Act
paternity index 106, 110-11
paternity testing 1, 105-11
absorption-inhibition ABO typing 2
lineage markers 131
PCR see polymerase chain reaction
peak balance 69, 70
personal objects 112, 129
phenol-chloroform-based DNA extraction 29,
phosphodiester bonds 8
PicoGreen ®  35
Pitchfork, Colin 1
Police and Criminal Evidence Act (PACE) 98
polymerase chain reaction (PCR) 39-50
components 40-2
contamination 45-8
DNA extraction 30
DNA quantification 32, 35-6
DNA replication 40
historical material 3-4, 39-40
hybridization-based methods 35
inhibition 44-5
laboratory set-up 46-8
lineage markers 126
low copy number 44, 68-70, 73, 101-2
post-PCR 47-8
pre-PCR 46-7
primers 41-2
procedure 42-4
reagents and buffers 42
real-time 35-6
sensitivity 45-6
Taq polymerase 40-1, 42-5
template DNA 40
see also short tandem repeats
polymorphic information content 138
population genetics see Hardy-Weinberg
positive paternity statements 109
post-PCR 47-8
power of discrimination 137
power of exclusion 137-8
PowerPlex ®  16 system
kinship testing 105, 109, 120
short tandem repeats 52, 61
PowerPlex ®  Y system 131
pre-PCR 46-7
presentation of evidence see evaluation process
presumptive testing 21-3
primers 41-2, 53
extension 36, 118-19
prior odds 92, 110-11
probe hybridization 3
ceiling principle 82, 83
frequencies 79, 88, 90
Profiler Plus ®  72, 106
prosecutor’s fallacy 91, 93-5
prostate specific antigen (PSA) 22
protein markers 2
proteinase K 30, 31
PSA see prostate specific antigen
pull-up 67-8
Punnett squares 75-6, 106-7
Quadraplex (QUAD) 52
Quantiblot ®  35
random match probabilities 88-9
random mating 77
re-offenders 97
real-time polymerase chain reaction (RT-PCR)
reference samples 1-2, 23
regulatory sequence 9-10
repetitive DNA 10
digestion 116
enzymes 2-3
Romanov royal family 128
RT-PCR see real-time polymerase chain reaction
saliva 17-19, 22
salting-out procedure 28-9, 31, 36
samples see biological samples
sampling bias 80, 81, 83
Sanger sequencing 2, 116-17, 118, 126
seminal fluid
biological samples 3, 17-19, 21-2
DNA extraction 30
DNA quantification 32-3
sex chromosomes see X chromosome; Y
SGM 52, 99
SGM Plus ®
evaluation process 88-9
national DNA databases 99, 101
short tandem repeats 52, 59, 61, 70
short interspersed elements (SINEs) 10
short tandem repeats (STRs) 4, 51-85
allele frequency databases 78-83
allelic ladders 58-61
corrections 78-83
degraded DNA 71-3
detection of polymorphisms 54-5
DNA structure 13, 14
drop-out 68, 69, 73
evaluation process 92
Hardy-Weinberg equilibrium 75-83
internal-lane size standards 56-8
interpretation 56-61, 65-74
kinship testing 111
lineage markers 130-2
low copy number DNA 68-70, 73
mixed profiles 70-1
multiplex development 51-3
national DNA databases 99
overloaded profiles 68
peak balance 69, 70
population genetics 75-83
primers 53
pull-up 67-8
single nucleotide polymorphisms 120-1
split peaks 65-7
standardization 52
structure 51
stutter peaks 65, 69
template DNA 68
silica based DNA extraction 28-9, 31, 36
silver staining 54
SINEs see short interspersed elements
single nucleotide polymorphisms (SNPs)
allele specific hybridization 119
detection 115-19
DNA structure 13-14
forensic applications 117-20
forensic identification 119-20
geographical ancestry 120
historical material 4
kinship testing 111
lineage markers 126, 130
occurrence and structure 115
primer extension 118-19
Sanger sequencing 116-17, 118, 126
short tandem repeats 120-1
SNaPshotTM 118
SNPs see single nucleotide polymorphisms
Southern blotting 2-3, 57-8
split peaks 65-7
likelihood ratios 91
match probability 88-9
positive paternity 109
uniqueness 89
storage of biological samples 23-4
STRs see short tandem repeats
stutter peaks 65, 69
allele frequency databases 80-2, 83
kinship testing 111
SYBR ®  Green 36
tandem repeats 10, 12-13, 14
see also short tandem repeats; variable
number tandem repeats
Taq polymerase
polymerase chain reaction 36, 40-1,
short tandem repeats 65-7
single nucleotide polymorphisms 117,
TaqMan ®  system 36
template DNA 40, 68
terminal transferase 66-7
tetranucleotide repeats 66
theta values 81-2, 83
transposed conditional 91, 93-5
ultraviolet spectrophotometry 34
uniqueness statements 89
United Kingdom NDNAD 97-102
cold cases 101-2
entry criteria 98-9
familial searching 100-1
legislation 98
operation 99-100
rationale 97
technology 99
variable number tandem repeats (VTNRs) 2-3,
evaluation process 92
lineage markers 130
national DNA databases 99
polymerase chain reaction 39
verbal scales 91-2
VTNRs see variable number tandem repeats
Yfiler ®  131
Watson, J. 7
X chromosome
amelogenin locus 53
structure 9
Y chromosome
amelogenin locus 53
lineage markers 125, 130-2
structure 9

Plate 3.1 Blood is the most common form of biological material that is recovered from crime scenes. (a) Large volumes of blood can be collected using a swab, if the blood is liquid then a syringe or pipette can be used (picture provided by Allan Scott, University of Central Lancashire) (b) Blood on clothing is normally collected by swabbing, or cutting out the stain (picture provided by Elizabeth Wilson)

Plate 6.4 During electrophoresis an argon laser is shone through the window in the capillary. As the labelled PCR products migrate through the gel towards the anode they are separated based on their size. When the laser hits the fluorescent label on the PCR products, the lable is excited and emits fluorescent light that passes though a filter to remove any background noise, and then on to a charged coupled device camera that detects the wavelength of the light and sends the information to a computer where software records the profile

Plate 6.5 The application of a matrix file, using the GeneScan® or GeneMapperTM  ID software removes the spectral overlap from the raw data (a) to produce peaks within the profile that are composed of only one colour (b)

Plate 6.6 Internal-lane size standards are used to size the PCR products precisely. Two commonly used internal-lane size standards are (a) the GeneScan®-500 (Applied Biosystems) and (b) the ILS600 (Promega)

Plate 6.9 The allelic ladder of the AmpFlSTR® SGM Plus® kit contains all the common alleles

Plate 6.11 The Genotyper® software compares the peaks within a profile to the allelic ladder and assigns alleles. If the peaks in the profile deviate more than ±0.5 bp from the allelic ladder they are designate ‘off ladder’

Plate 7.5 If the reaction is overloaded with DNA, (a) the peaks are still present but artefacts such as pull-ups and split peaks are more pronounced. When the template is within the optimal range (b and c) the peaks are well balanced and easy to interpret. When the PCR does not have enough template to amplify (d), then locus and allelic drop-out can occur

Plate 7.6 A heavily overloaded profile. All the peaks shown have a flat top indicating that they are off-scale, the baseline is very noisy, several split peaks are evident and the peaks are very broad, which can lead to sizing problems. There are also some pronounced stutter peaks

Plate 7.10 The profile was generated using the AmpFlSTR® Profiler Plus® kit from Applied Biosys-tems. The DNA was extracted from a bone recovered from a Scottish loch after approximately 30 years. The profile is typical of a degraded profile with a gradual reduction in the amount of product as the amplicons increase in size

Plate 7.11 The profile was generated using the AmpFlSTR BlueTM kit from Applied Biosystems. The DNA was extracted from muscle tissue recovered from a plane crash. The muscle had been subjected to high temperatures and the DNA was highly degraded – no amplification products were detected from the FGA locus. The size standard is also shown in by non-shaded peaks

Plate 11.1 A STR profile generated from foetal cells recovered from amniotic fluid in early pregnancy. In this case it was possible to determine that pregnancy had resulted from a rape, and allowed an informed decision to be made on whether or not to have the foetus aborted. The profile was generated using the AmpFISTR® Profiler Plus® STR kit (Applied Biosystems)

Plate 11.2 The identification of human remains recovered from an air crash [35]. Blood samples were provided by the mother and father who were missing a son. Alleles in the profile of human remains could have come from the mother and father (indicated by the arrows). The profiles were generated using the AmpFISTR® Profiler Plus® STR kit (Applied Biosystems)

Plate 12.2 A primer anneals to the template strand. This is extended by Taq polymerase until a ddNTP is incorporated. The ddNTPs are incorporated at random, which leads to a collection of extension molecules that differ from each other by one nucleotide (shown above labelled 1 to 8). The four ddNTPs are labelled with different fluorescent dyes that are detected during capillary electrophoresis

Plate 12.3 The sequence of a region of the mitochondrial genome. The sequencing software inter-prets the sequence data and ‘calls’ the bases. This information is provided above the sequencing peaks

Plate 12.4 The primer extension assay. (a) The target sequence is amplified using PCR and the products are used as the template in the extension assay; (b) an internal primer hybridizes to the target adjacent to the SNP and a single fluorescently labelled ddNTP is added by Taq polymerase; (c) the reaction is analysed by capillary electrophoresis

Plate 13.3 The above sequence shows the presence of heteroplasmy at position 16189. Two bases, a Cand an A, are presentin approximately equal amounts

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