Tags & Description
DNA
deoxyribonucleic acid -nucleic acid (polymer) -macromolecule
central dogma
DNA is transcribed to RNA; RNA is translated to proteins DNA --> RNA --> proteina
nuclear DNA (nDNA)
found in the nucleus
Mitochondrial DNA (mtDNA)
found in the mitochondria
supercoiling
DNA is packed by this process, wound tightly around histones to form nucleosomes
histones
offer level of protection to DNA
chromatin
densely packed DNA found in the nucleolus of nucleus
human karyotype
consists of 22 matched pairs of autosomes (non-sex cells) and a pair of two sex chromosomes (XX-F and XY-M)
genome
complete set of instructions for making an organism (entire DNA within cell), consists of DNA in all of its chromosomes
functions of genome
-complete set of genetic information -includes coding DNA (exons- extrovert/express) -non-coding DNA (introns- introvert/no express)
genes
genetic information that is coded and packaged
genotypes
set of genes
phenotypes
observable characteristics
hybridization
DNA is linked together
nucleotide
individual unit (building block) of DNA, pair with complementary base via hydrogen bonds (A --> T (U) double bond) (G --> C triple bond)
nucleotide components
-pentose sugar (deoxyribose in DNA, ribose in RNA) -nitrogenous base (Adenine, Guanine, Cytosine, Thymine-DNA ; Adenine, Guanine, Cytosine, Uracil- RNA) -phosphate group
nucleoside components
-nitrogenous base -pentose sugar **bases and pentose sugars are heterocyclic compounds
Nitrogenous base
4 possibilities at each position (A, G, C, or T)
trillions of combinations are possible
** the info content in the DNA is encoded in the order (sequence) of the bases ** ex: cats --> scat, snn, cell #
dATP
deoxyadenosine 5'-triphosphate or deoxyadenylate
gametes
or germ cells (egg or sperm) have haploid or single set of chromosomes
somatic: body cells
any cell other than germ cell
haploid cells
3 picograms, one set of chromosomes, n
diploid cells
6 picograms, two sets of chromosomes, 2n
RNA
carries messages encoded in DNA sequence to the cytoplasm (specifically mRNA) since DNA cannot leave nucleus, proteins are translated in cytoplasm
forms of RNA
-ribosomal RNA (rRNA) -messenger RNA (mRNA) -transfer RNA (tRNA) -microRNA (miRNA)
microRNA
(miRNA) small non-coding RNA and can be used for body fluid identification (tissue specific), HTS (sequencing) of miRNA of interest and comparison to reference
purine
Adenine and Guanine have a double-ring structure with six-carbon ring fused to five-carbon ring
pyrimidine
Cytosine and Thymine smaller bases that only have a six-carbon ring structure
gene locus
specific location on a chromosome where a coding region exists (in forensic science look at non-coding region) singular- locus plural- loci
allele
form of the gene locus or an alternate form of a gene*** ***bolded in notes
homozygote
two copies of the same allele or form of the gene locus, individual has two alleles of the name # of repeats, both alleles same length
heterozygote
different alleles or forms of the gene locus, individual has two alleles with different # of repeats, alleles differ and can be resolved from one another
STR
short tandem repeat regions of DNA that are repeated (typically in non-coding region) single stranded DNA
locus
specific STR marker
Restriction Fragment Length Polymorphism
(RFLP) early technology, required a lot of DNA and high quality DNA to work (started with this analysis technology) Dr. Alex Jeffries ?
HLA DQA1
(reverse dot blot SNP assay) first PCR-based technology, the later addition of Polymarker (PM) made the technique more discriminatory
STR analysis
more discriminatory, faster, requires less DNA, can analyze degraded samples; autosomal
challenges of DNA rape case
-mixtures must be resolved -DNA is often degraded -inhibitors to PCR (polymerase chain reaction) are often present- heme can be an inhibitor
forensic cases
matching suspect with evidence
paternity testing
identifying father
mass disasters
putting pieces back together
human identity testing
-forensic cases -paternity testing -historical investigations -missing persons investigations -mass disasters -military DNA "dog tag" -convicted felon DNA databases
Y-STR analysis
Y chromosome, number of male contributors, paternal inheritance
mtDNA features
shape: circular genetic alphabet: 16,569 base pairs copies per cell: 100's-1000's inherited: 100% mother location in cell: mitochondrion unique: no
nucDNA features
shape: linear genetic alphabet: ~3 billion base pairs copies per cell: 2 inherited: 50% mother, 50% father location in cell: nucleus unique: yes
nuclear and mtDNA specimen type
-blood -tissue -hair (w/ root) -fresh bone/teeth -body fluids -stamps/envelopes
mtDNA only specimen types
-skeletal remains (not flesh) -hair shafts -fingernails
steps in DNA (STR) analysis
extraction
quantification
amplification
separation
analysis and interpretation
report conclusions (and statistics) ADD CHART
extraction
isolate DNA
quantification
how much DNA is in extract
amplification
make many copies of DNA sample (at different STR markers) for analysis, polymerase chain reaction is used
separation
(capillary electrophoresis) separate DNA fragments
interpretation
determine DNA profile of a sample by analyzing electropherogram from the Capillary Electrophoresis
statistics
IF MATCH, is there reference to profile and/or database
Human nuclear DNA is not found in which of what cell type?
red blood cells
impurities
-protein -ionic species -compounds -cell debris -carbohydrate -internal cellular structures
steps for isolation of DNA
open organism/ cell that contains DNA
separation of DNA from other cellular components
organism/cell with DNA
-any human biological specimen -non-human biological specimen -plants, seeds, leaves -microbial specimen (terrorism)
cellular components
remove inhibitors
heme from blood
humic acid, fulvic acid, calcium and collagen (bone samples)
melanin in hair, skin
inhibitors can inhibit enzyme polymerase, or Mg
chelex
simple system for neat samples Ex: saliva reference samples compound you use 1 tube, extract DNA from same tube
FTA
card based extraction system
robotic
simple, magnetic bead extraction technique -Dr. Roy uses for her research
organic
more challenging samples, gets rid of impurities Ex: blood, etc gold standard, get double stranded DNA
differential extraction
complex samples with possible mixture, used in sexual assault cases (separate sperm from other cells)
solid-phase extraction method
qiagen
chelex-100
ion-exchange column resin, iminodiacetic acid
binds magnesium and removes them from the reaction mixture = DNA stabilized and preserved, extracted DNA is partially single stranded (due to heat)
metal ions Zn, Mg, Ca, can act as catalysts or cofactors for nucleases and thus help degrade DNA by enzymatic degradation of hydrolysis
iminoacetate ion
chelating characteristics
chelating
binding of ions or molecules to metal ions (gives single stranded DNA)
RFLP
double stranded DNA
reagents in differential extraction
lysis buffer
SDS (Sodium dodecyl sulfate)
Pro K (Proteinase K)
DTT (Dithiothreitol)
EDTA (Ethylenediaminetetraacetic acid)
buffer/ions
Lysis buffer
chemicals and proteins added to the sample that facilitate lysis of the cell membrane and effective removal of impurities
SDS
sodium dodecyl sulfate (detergent)
why shampoo forms
Pro K
proteinase K (digests proteins and chops it up)
need to get rid of proteins
DTT
dithiothreitol (differential extraction, breaks the S-S bonds and allows the protein to break apart)
sperm heads broken down
EDTA
ethylenediaminetetraacetic acid (chelates metal ions that can degrade DNA and cause hydrolysis of DNA molecule
acts like chelex
buffer/ions in differential extraction
maintains pH, ions helps later in the downstream Ex: Tris-HCl and NaCl
basic rudimentary DNA
quantitative PCR (qPCR)
real time PCR determine the amount (quantity) of DNA present in the extract to assist in determining the appropriate amount of the extract to add for amplification
thermal cycler
polymerase chain reaction (PCR) is performed by this, repeated cycles of heating and cooling like a copy machine
steps of PCR
denature
anneal
extend DAE
PCR reagents
template DNA (single stranded)
short primers (initiate synthesis of new DNA)
dNTPs (add to growing strand of DNA)
polymerase (perform synthesis of new DNA)
MgCl2 (activate polymerase)
Old Faithful w/ hot --> cold --> hot
DNA amplification steps
starting DNA template (heat needed to start first step)
denaturation, double stranded DNA dissociates
separate strands (denature)
binding of the primer to provide initiation site for DNA synthesis
add primers (anneal)
extension of DNA from primers
new strand synthesis (extend)
multiplex PCR
started with 10 markers
over 26 markers can be copied at once
sensitivities to levels less than 1 ng of DNA
ability to handle mixtures and degraded samples
different fluorescent dyes used to distinguish STR alleles with overlapping size ranges
separation by capillary electrophoresis (CE)
like liquid gel electrophoresis
separation of fragments by size using applied electric voltage
DNA is negatively charged = small fragments move towards positive charge faster than larger fragments fragments tagged with dyes, captured by camera as they migrate instrument: 3130 xL genetic analyzer
TH01
human tyrosine hydroxylase gene 01-repeat region is located within intron 1 of tyrosine hydroxylase gene
HUM
human genome prefix
STR locus TH01
HUMTH01
gene name
-IF marker WITHIN gene or PART of gene, gene name used -marker OUTSIDE gene regions, designated by CHROMOSOMAL position
example of locus not w/in gene regions
D5S818 D- DNA 5- chromosome 5 S- DNA marker is a single copy sequence *S represents unique DNA segment 818- order of discovery/ order in which locus was identified **ALL OF THE ABOVE MAKES LOCUS UNIQUE
DYS19 D- DNA Y- y chromosome s- single copy sequence 19- order discovered
do we need to add DTT (dithiothreitol) to non-sperm samples?
no, do not need to be broken?
single source
typically for each locus, expect to see 2 alleles ( 2 peaks)
2 alleles ~ heterozygote -1 allele ~ homozygote
mixture
when more than 2 alleles are see at two or more loci contributors- 3 peaks - 2 cont., 5 peaks - 3 cont., 6 peaks - 3 cont., 7 peaks - 4 cont., 8 peaks - 4 cont.
amelogenin
sex determining locus gene on the x-chromosome that codes for proteins associated with tooth enamel
x-homologous amelogenin gene region
exists on y-chromosome, region has 6 bp deletion, so x and y products differ in size
factors that can complicate interpretation
stutter
pull up
spikes
dye blobs
degradation
low DNA template amount (can lead to stochastic-random effects, drop out)
stutter
during PCR DNA is improperly replicated so one repeat is shorter than parent strand, slipped strand mispairing model come together out of sync, shows up as little peaks
conclusion
made after comparing a profile to a reference
inclusion ** (must always provide statistics)
exclusion
inconclusive
inclusion
evidence profile and reference profile appear to match weight of the match must be reported in conclusion via statistic, how strong of a reference profile to evidence profile
single source profiles
random match probability (RMP) weight between single source and evidence court order STRONGEST STATISTIC TO USE
mixture profiles
modified random match probability (mRMP)
combined probability of exclusion (CPE)
combined probability of inclusion (CPI)
likelihood ratio (LR)* - becoming very popular
CODIS
combined DNA index system, represents the software software stores data, interpol can see w/ permission to look
used for linking serial crimes and unsolved cases w/ repeat offenders at local, state and national level
US national DNA database
launched oct 1998
links all 50 states
when started required >4 RFLP markers
currently 20 core LOCI (STR) markers, STR core loci at the beginning started with 13