DNA last 2 lectures

Multiple depositors, low-level contributor, secondary transfer

Name the different types of mixture DNA

_______ ___________: Two or more individuals physically deposit biological material at the same location

___-_____ __________: One contributor is present at a very low quantity, difficult to detect

_________ ________: DNA is indirectly deposited require an intermediate surface

Not all contributors are equally visible

Numbers of contributors, contributor ratio, allele sharing, and total DNA quantity

What are factors that affect mixture detection?

D18S51

What is the high diversity locus that makes the most informative markers for mixture interpretation (about 50 possible alleles)?

Major contributor

Individuals who contributed more DNA

Minor contributor

individuals who contributed less DNA

Mixture ratio

Estimate the mixture ratio - the relative amount of DNA each person contributed

Large differences in peak height = Larger major : Minor Ratio

5:1 Noticeably smaller peaks present- causes minor still detectable interpretation

10-20:1 some minor peaks missing - causes allele dropout

Detection depends on Peak height (RFU thresholds)

If you do not see the minor contributor, it does not mean they are not there.

70

Small peak / Large Peak > __% indicates a single source profile

if the ratio is below __%, indicates mixture; degradation; Inhibition

10-15

Normal stutter peak: small; <__-__% of the main peak; Always appears in a predicable position (n-1)

Mixture peak: Looks like stutter but larger

Not all small peaks are stutter artifacts.

Stutter can hide or mimic minor contributors, making mixture interpretation more difficult

Steps approach to mixture interpretation

1. Is it a mixture? more than 2 peaks at a locus; big peak height ratio; Unexpected allele patterns

2. Label the peaks: above analytical threshold

3. How Many contributors. Max alleles / 2 = minimum contributors

4. Who is Major and Minor, Tall peaks and shorter peaks

5. What genotype combination make sense

6. Compare to known profiles

Calculating mixture ratio

Major contributor (B+D)

Minor contributor (A+C)

Ratio = (B+D) / (A+C) = 2.3 : 1

Round to approximately 2:1 - The major contributor contributes roughly twice the DNA of the minor contributor

Allele sharing, more than two contributors, low template DNA, stochastic effects, degradation, and inhibition

What are some challenges in Mixture interpretation?

Probabilistic genotyping

What type of interpretation method is used by software like STRmix to analyze complex DNA mixtures?

STRmix

This software is important because it uses real forensic labs, accepted in courts and helps analyze mixtures that are too complex to interpret manually

It tests millions of possible genotype combinations and uses the entire electropherogram. Accounts for peak height, dropout and stutter

Preforms interpretations faster, more consistently and with statistics

Single nucleotide polymorphisms (SNP)

A single base change in DNA occurs at a specific location

There are so many of them

Advantages:

• Extreme Abundance: every 1000 bp

• Genome-Wide Coverage - located across all chromosomes

• Give a big picture of DNA

• Especially useful when DNA is damaged or limited

Disadvantages:

• Low power per marker

• Need many SNPs, 50-100+ markers; more cost

• Mixtures are Harder- Limited variation and difficult to separate contributors

• Limited Databases

Technology Enables: Next-generation sequencing (NGS) panels

Reading results:
Each peak = a base (ATCG), each color = a different base

One peak = homozygous; Two Peaks = Heterozygous

STR vs SNP

Fill in the blank…

___: Repeat length, Often 10+ per locus, high distinguish power, length (100-400+ bp), stutter artifacts: present complicates interpretation. Databases: CODIS, NDNAD

___: Single nucleotide substitution. only 2 bi-allelic, Lower discrimination power on one locus, Very small (<100bp), no stutter artifacts, clear signal, No standard forensic databases.

Direct sequencing, primer extension (SNaPshot)

This SNP method reads the actual DNA sequence. Use next-generation sequencing (NGS).

The other common SNP method is ______ _______ (_______) that targets specific SNPs. Targets a single base using fluorescently labeled dNTPs.

Less commonly used: Microarrays and pyrosequencing

SNaPshot

A method to identify SNP alleles and looks at one base in DNA

DNA region is amplified (PCR), primer binds next to the SNP, One base is added (ddNTP) and that base is fluorescently labeled.

Each base=different color

Reads only one base. Color = SNP allele

Protocol:

PCR - Clean up extra primers and ddNTP - Extension add one base - electrophoresis (read color)

Massively parallel sequencing (MPS)

What type of sequencing technology analyzes thousands to millions of fragments at once?

Ancestry informative markers (AIMS)

What are specific single-nucleotide polymorphisms (SNPs) that show significantly different frequencies between human populations, making them powerful tools for estimating biogeographical ancestry.

Internal size standard

What is used during capillary electrophoresis to ensure accurate sizing of DNA fragments?

Amalyase

What enzyme is commonly used to identify saliva?

Acid phosphatase (AP)

What enzyme is targeted in presumptive tests for semen?

Test for contamination

What is the purpose of an extraction control (reagent blank)?

variable number tandem repeats (VNTR)

What type of repeated DNA sequence was used in early DNA fingerprinting before STR?

Microvariant

Which allele call contains an incomplete repeat (eg 9.3)

Normalization

What is it called when you adjust DNA input volume to ensure consistent amplification across samples?

Threshold cycle (Ct)

What value in qPCR represents the cycle at which fluorescence exceeds the detection threshold?

Low DNA level

What does a high Ct value indicate about a DNA sample?

(hint: it took longer for sample to hit the threshold)

Stutter

What is the most common biological artifact in STR analysis caused by strand slippage?

Proteinase K

What enzyme is used to breakdown proteins during DNA extraction?

Measure inhibition

What is the purpose of an internal positive control (IPC) in qPCR? (quantitation)

Linkage equillibrium

What conditions must exist between loci to justify multiplying probabilities across STR markers?

Porous

What type of surface may initially capture more DNA but may make recovery hard?

Solid phase extraction

DNA extraction method that binds DNA to solid surface like silica or magnetic beads

What happens if too much DNA is added to PCR reaction

Overamplification and split peaks

DTT

What is added to break disulfide bonds in sperm cells during differential extraction?

Product rule

Which rule allows forensic scientists to multiply genotype frequencies?

Background DNA

What is the term for DNA already present at the crime scene?

RMP

What statistical value represents how rare a DNA profile is within a population?

Pull-up

What artifact occurs when signal from one dye appears in another color channel?

Analytical

Which threshold separates true signal from background noise in STR analysis?

Heat stable

Why is Taq polymerase used in PCR instead of regular DNA polymerase?

Tetranucleotide

Which STR is preferred in forensic science due to low stutter?

GeneMapper

What software is commonly used for STR genotyping and data interpretation?

Dye-labeled primer

This type of primer is useful in STR analysis as it separates different fragment sizes for a more clear result.

Denaturing

What type of electrophoresis conditions keep DNA as single strands for better resolution?

Null allele

What is the name for an allele that fails to amplify due to a primer binding size mutation?

Fragmented (degraded)

What is it called when DNA has been broken into smaller pieces?

Stacking

What is the term for when two contributors share the same allele causing peaks to combine?

DNA sequence

What does next generation sequencing (NGS) measure that STR analysis does not?

Prosecution / defense

The likelihood ratio (LR) includes two hypothesis. What are they (in order)?

Theta (θ)

What is the statistical adjustment when there is not enough DNA data for population genotype frequencies? (hint: 0.3)

Both hypothesis are equally likely

What does a LR=1 indicate?