Quiz 4

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

of repeats exhibited in a particular STR marker, resulting in different length

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

1. extraction

2. quantification

3. amplification

4. separation

5. analysis and interpretation

6. 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

1. open organism/ cell that contains DNA

2. 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

1. starting DNA template (heat needed to start first step)

   • denaturation, double stranded DNA dissociates

2. separate strands (denature)

   • binding of the primer to provide initiation site for DNA synthesis

3. add primers (anneal)

   • extension of DNA from primers

4. 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