exam 2 terms for forensic biology

quantification is used to determine BLANK

how much DNA has been extracted from sampeles

high levels of DNA create BLANK

more artifact peaks

low levels of DNA produce BLANK

allele dropout

spectrophotometers measures BLANK

absorbance of light at specific wavelengths

absorbance of nucleic acid (DNA, RNA, nucleotides)

260nm

absorbances of protein

280nm

absorbance of guanidine, phenol, salts

230nm

pure DNA and RNA have A₂₆₀/A₂₈₀ ratios of BLANK

1.8 and 2.0

A₂₆₀/A₂₈₀ ratios <1.7 indicate BLANK

protein contamination

pure DNA and RNA have A₂₆₀/A₂₃₀ ratio between BLANK

2.0-2.4

anything outside the stated A₂₆₀/A₂₃₀ ratio range means BLANK

contamination with extraction chemicals

absorbance readings give an estimation of purity, they do NOT indicate BLANK

DNA or RNA integrity

fluorometers use a BLANK

light source to excite specific molecules and then detect the amount of fluorescence emitted

PicoGreen dsDNA quantification

ultra sensitive fluorescent nucleic acid stain for DNA quantification

Quibit Fluorometer

highly selective for targets bound to fluorescent molecules

parts of fluorescence

10,000 fold more sensitive that UV detection

highly selective to dsDNA

little interference from ssDNA and RNA

fluorometer or fluorescent plate reader

no assessment of purity

parts of UV absorbance

relative insensitivity of the assay detection range

interference in signal level from contaminants such as RNA, nucleotides, proteins, salts, phenol, etc

UV spectrophotometer

no standards needed

assess purity

polymerase chain reaction (PCR) was devised in 1983 by BLANK

Kary Mullis

PCR enables BLANK

vast quantities of specific DNA to be produced quickly and cheaply

PCR solves the problem of BLANK 

how to make copies of a targeted region of DNA

PCR uses the same biomolecules that cells use for DNA replication

2 primers

thermostable DNA polymerase

nucleotides (A,T,G,C)

3 steps of PCR cycle

1, denaturation

2. annealing

3. extension

PCR amplifies DNA BLANK

exponentially

PCR components

buffer solution

Mg²⁺ ions

dNTPs

primers

thermostable DNA polymerase

DNA template

buffer solution

creates optimal conditions (including pH) for polymerase activity

Mg²⁺ ions

critical for Taq DNA polymerase activity

dNTPs

building blocks of newly synthesized DNA molecules

primers

short synthetic pieces of DNA and define initiation site for elongation of new DNA

thermostable DNA polymerase

normally Taq DNA, catalyzes synthesis of new DNA

DNA template

initial strand that Taq builds from

primers anneal to template DNA and direct Taq polymerase to move BLANK

5’ to 3’

high primer concentration =

off-target amplification and primer dimer formation

low primer concentration =

alter PCR efficiency

BLANK is the typical choice for PCR

Taq DNA polymerase

purpose of Hot Start

to without a crucial reaction component (ex. Taq DNA polymerase activity) until the first cycle rises above annealing temperatures

BLANK was developed by Higuchi and colleagues in 1992

qPCR (“real time” PCR)

qPCR allows the detection of the amplicon as it BLANK

accumulates during PCR cycles

types of fluorescent chemistries available

DNA binding dyes (SYBR Green I) (not sequence specific)

Hydrolysis (TaqMan) probe (sequence specific)

4 phases of qPCR amplification

1. lag (baseline)

2. exponential

3. linear

4. plateau

lag (baseline)

too low to be distinguished from background noise

exponential

100% efficiency, exact doubling of PCR, specific and precise (signal become detectable)

linear

highly variable, reaction components consumed, linear amp rather than exponential

plateau

end point analysis, components have been consumed, if left too long could degrade PCR product

the cycle threshold (CT) is set a BLANK

particular fluorescence value in relative fluorescence units (RFUs)

each sample is given a BLANK

CT value

SYBR Green I is a BLANK

DNA binding dye (intercalates DNA)

features of SYBR Green I

not human specific 

binds to dsDNA

Hydrolysis (TaqMan) probes use

2 primers and a sequence-specific oligonucleotide (TaqMan) probe

parts of TaqMan probe

reporter dye attaches to 5’ end

quencher attached to 3’ end

when probe is intact fluorescence for reported is quenched

during extension annealed probe is cleaved by Taq’s 5’-3’ exonuclease activity

reporter separated from quencher =

fluorescence

positives of TaqMan probe

highly specific

non specific amplicons DON’T affect quantifications accuracy

multiplex possible

higher sensitivity

negatives of TaqMan probe

requires optimization

more complex assay design

slower because of optimization

higher costs

positives SYBR Green I

little optimization required

simple assay design

multiple genes can be tested quickly

low initial cost

negatives of SYBR Green I

lacks specificity, binds to any dsDNA

non specific amplicons DO affect the accuracy of quantification

cannot multiplex

lower sensitivity

thermal cycles of qPCR

specialized thermal cycles must be used to measure the fluorescence during each PCR cycle

Quantifiler Trio kit is used for BLANK

qPCR

Quantifiler Trio kit provides BLANK

quantitative and qualitative assessment of total human or human male DNA in a single, highly sensitive real-time PCR

Quantifiler trio kit includes

large autosomal target

small autosomal target

Y-chromosomal target

IPC
NTC

internal positive control (IPC) is a BLANK

short strand of synthetic DNA that is amplified in the same reaction of your DNA samples

IPC is used to assess the BLANK

quality of your sample and confirm that components of the assay are functioning properly

IPC amplifies, but sample does not =

low amount/no starting template

IPC DNA and sample do not amplify =

inhibition

NTC

no template control (AKA no DNA)

can compare CT of the IPC and CT of the NTC to check for BLANK

inhibition

DNA degradation is assessed by comparing the BLANK

ratio of large target amplification to small target amplification in the assay

if the large target is degraded =

there is less amplification

degradation index (DI) equation

small autosomal target concentration / large autosomal target concentration

BLANK are helpful in sexual assault kits and mixtures

mixture assessments

ratio of autosomal DNA detected compared to Y-chromosome DNA detected can indicate if BLANK

a male/female mixture is present

mixture ratio format

male DNA: female DNA

female DNA equation

total human DNA conc - total male DNA conc

stages of DNA profiling

extraction

quantitation

amplification

fragment separation

analysis

extraction

cells are collected, disrupted to release, and isolate small amounts of DNA

quantitation

DNA is quantified

amplification

multiple STR regions of DNA are amplified by PCR

fragment separation

PCR products are sized using capillary electrophoresis

analysis

pattern of fragment distribution is analyzed/compare

minisatelittes/VNTRs =

repeated sequence 6-100 bp

microsatellites/STRs =

repeated sequence 1-5 bp

features of STRs

mono, di, tri, tetra, penta, or hexa repeats

smaller size is easier to accurately amplify by PCR, even for degraded DNA

high heterozygosity for discrimination power

locus =

singular

loci =

plural

forensic STR markers focus on BLANK

tetranucleotide repeats (some pentanucleotide markers)

positives of tetranucleotides

low stutter 

fewer undesirable PCR products

core 13 loci was established in BLANK

1997

CODIS

combined DNA Index System database

core 20 loci was effective as of BLANK

January 1st, 2017

advantages of 

>100,000 STRs identified in human genome

multiple alleles

highly heterozygous

tetra or pentanucleotides

found in different chromosomes or far enough apart that they are in linkage equilibrium

small size 100-400 bp

relatively low mutation rate

ability to be co-amplified/multiplexed

non-coding (intronic)

side note for following :

• multiple alleles

• highly heterozygous

DNA profile will be highly discriminating

side note for following : 

• tetra or pentanucleotides

produce less stutter

side note for following : 

• found in different chromosomes or far enough apart that they are in linkage equilibrium

inheritance of alleles is independent

side note for following : 

• small size 100-400 bp

amenable to amplification with PCR

side note for following : 

• relatively low mutation rate

easily assess the frequency of alleles

side note for following : 

• ability to be co-amplified/multiplexed

looks at lots of alleles at once (save time, money, sample0

side note for following : 

• non-coding (intronic)

genetic privacy

things in STR kits

PCR primer mixture (primers labeled with fluorescent dyes)

PCR buffer including all reagents for PCR reaction

DNA polymerase

allelic ladder with all common alleles of STRs included to calibrate allele repeat size

positive control DNA to ensure reagents are functioning properly

labs use PCR systems that examine BLANK

multiple STR loci in a single reaction

using multiple STRs increases the BLANK

power of discrimination

major suppliers

Applied Biosystems (AB)

Promega

Qiagen

kits approved for use with NDIS that contain 20 core loci:

AB Globalfiler

Promega PowerPlex Fusion

Qiagen Investigator