FOR BIO midterm

ch 1-7

Alphonse Bertillon

working for the police in Paris in the late 1800’s, he had a profound impact on the development of criminal investigations

Anthropometry (Bertillonage) to identify criminals

 Incorporated facial photographs

 Crime scene documentation

Dr. Edmond Locard

student of Bertillon who made extensive use of microscopy  director of the first crime laboratory, in Lyon, France

Locard observed that criminals could be associated with particular locations, items of evidence and victims. “When any person comes into contact with an object or another person, a cross-transfer of physical evidence occurs“

pcr

polymerase chain reaction

what inhibits pcr assay

hemoglobin,

urea, heparin

organic/phenolic compounds

glycogen,

fats, Ca 2+

tissue matrix effects: laboratory items, powder

PCR enhancers

DMSO, glycerol, BSA

Formamide, PRG, TMANO, TMAC

special commercial enhancers: gene 32 protein, perfect match, TAQ extender, e.coli ss DNA binding

Kastle-Meyer test

presumptive blood test (Phenolphthalein)

• dry sample collected w/ swab/filter paper

• few drops of alcohol, then a few drops of phenolphthalein and finally a few drops of hydrogen peroxide are dripped onto the sample

• if sample turns pink then it is a positive test

• nondestructive to the sample; it can be kept and used in further tests at the lab

• same reaction with blood from any animal; further testing required to determine if it is human

Hemastix

presumptive blood test

- detects the peroxide-like activity or hemoglobin in a substance

process

• apply drop of DI water on tip of stick

• rub pad on suspected substance

• pad will turn from yellow to dark green depending on the amount of hemoglobin found

• – compare the reaction to the chart on the side of the bottle

Hematrace

presumptive blood test

testing device used to aid in the possible ID of human blood by detecting presence of human hemoglobin

if human hemoglobin is present in sample its antigens will react w/ mobile monoclonal antihuman complex that moves through device

Test has shown that false positives may occur with ferret blood

luminal

presumptive blood test

presumptive semen test

p30 (confirmatory testing)

Acid Phosphatase testing

alternate light source

stutter - errors in sequencing

Peaks that show up primarily one repeat less than the true allele as a result of replication or strand slippage during DNA synthesis.

• Stutter is less pronounced with larger repeat unit sizes (dinucleotides > tri > tetra > penta).

• Longer repeat regions generate more stutter.

• Each successive stutter product is less intense (allele > repeat-1 > repeat-2).

• Stutter peaks make mixture analysis more difficult

Allele dropout

PCR amplification may fail due to sequence changes in a primer binding region of genomic DNA template, making it impossible to detect an allele that exists in the template DNA. This results in what is known as null alleles.

Artifacts: Non-template A Addition

Terminal 3’ A that is often added to the growing PCR product changes allele size
Problem occurs when some fragments have A’s and others do not

It is possible to either force all fragments to have the A’s or to remove all the A’s

◼ Long extension step after PCR will promote the A addition

◼ Specific sequences (pigtail – GTTCTT) will promote the A addition

◼ Using too much DNA will lead to incomplete A addition

sample mixture

crime scene evidence sometimes mixed

Ideally, you want to have evidence that is not mixed and only use mixed STR profile data as a last resort. However, given the large number of alleles, the forensic analysis of mixed samples with STRs is possible. Often it will be possible to determine if a suspect is excluded as the source of the sample. In some cases, it is feasible to differentiate the individual components of the mixed sample.

unrestricted vs restricted

Unrestricted: ignores relative peak height differences

Restricted: based on relative peak heights

dealing with dna mixtures effects

Transfer of Matter

When two objects come into contact, traces of each are left on the other.

The first markers used in DNA forensic typing

RFLPs (Reconstruction Fragment length Polymorphisms).

When was the first polymorphic RFLP marker discovered and by who?

1980 by Ray White.

Who developed multi-locus RFLP probes?

Alec Jefferys in 1984.

RFLP method

Restriction enzymes cleave DNA at specific sites in a sequence dependent manner.

RFLP markers in forensics are characterized by what?

VNTRs (Variable Number of Tandem Repeats).

VNTRs

Polymorphisms in the length of tandemly repeated short sequences. 7-100 bases. Also known as minisatellites.

How many alleles are there per locus in VNTRs?

There are dozens per locus.

What is RFLP analysis used for?

To identify a specific DNA in a complex mixture of DNAs.

Steps of RFLP Analysis

1. Isolation of genetic material and PCR.
2. Restriction digestion of amplicons.
3. Electrophoresis of digested fragments.
4. Visualization.

Single-locus probes for VNTRs

Probes are complementary to sequences that are locus-specific. They hybridize to ONLY ONE LOCUS in the human genome.

Multi-locus probes for VNTRs

Probes are complementary to sequences that are present in different genomic regions. They hybridize MULTIPLE LOCI dispersed through the human genome.

What are VNTR probes?

Labeled oligonucleotides that are complementary to a given sequence present in the human genome.

What do multi-locus probes detect?

Multiple variable DNA fragments by Southern Blot to make an individualizing "DNA fingerprint".

How many loci is genotyping performed on? (VNTRs)

Several loci one at a time.

DNA fingerprinting vs DNA profiling. (RFLPs)

Fingerprinting is the RFLP method of using multi-locus probes.

Profiling uses single-locus probes.

Advantages and Disadvantages of RFLP.

Advantage: High power of discrimination.

Disadvantages:
- Need large amounts of sample.
- Sensitive to DNA degradation.
- Time-consuming.
- Technical problems for allele identification.

PCR vs RFLP Differences

PCR:
- Less DNA needed.
- 1-2 day turnover.
- Can use highly degraded DNA.
- Discrete alleles obtained.
- High-volume sample processing.
RFLP:
- 6-8wks radio./1wk chem. probes.
- More DNA needed.
- Needs intact DNA
- Cannot process high-volume samples.

PCR and RFLP Similarities

- Capable of handling sample mixtures.
- Allele identification.

Who discovered PCR and when?

Kary Mullis in 1983. Described it in 1985.

Polymerase Chain Reaction (PCR)

Minute amounts of DNA can be amplified into multiple copies.

Steps of PCR

1. Denaturation (94-96 C)
2. Annealing (55-68 C)
3. Extension (72 C)

When were the first PCR-based method introduced?

1990

What was the first PCR marker used?

DQA1, located within the HLA region of Chromosome 6.

Nucleotides and Bonds

Adenine=Thymine (2)
Cytosine=-Guanine (3)

A,T,C,G

Purines: Adenine, Guanine
Pyrimidines: Thymine, Cytosine

PCR primers consist of:

Two oligonucleotides hybridize to complementary strands of DNA template, identifying the region to be copied. 3' OH group NEEDED.

selecting Loci

Includes consideration of method development. Selected for suitability in a multiplex system and their discriminating power.

PCR Reagents:

Taq polymerase, dNTPs, Mg ion, PCR buffer (Tris-HCl), BSA, primers.

Primer Annealing

Phase in PCR during which a primer binds to a template strand. Dependent on base composition.

Primer Extension

DNA polymerase extends the primer by its polymerase activity. Done at 72C.

Plateau Effect

Attenuation in exponential rate of product accumulation.

Transcription vs Translation

Transcription: Gene expression.
Translation: Protein synthesis.

Reverse dot-blot method

13 probes on the Strip. Probes are used to genotype 5 different markers w/ 2-3 alleles.

What replaced Amplitype DQA1 + Polymarker?

STRs (Short Tandem Repeats) aka microsatellites.

Segregation

Separation of HOMOLOGOUS chromosomes into gamete cells.

Independent Assortment

NON-HOMOLOGOUS chromosomes can combine in many ways during meiosis.

Exceptions to Independent Law of Assortment

- Genetic markers found close together on same chromosome are transmitted as a block (linkage).
- Genes located close to each other show tight linkage.

What acts to reduce linkage?

Recombination

Flowchart of Forensic DNA Typing

Sample - Forensic Eval. - Cell/Diff. Lysis - Purification of DNA (Chelex/Organic/Silica) - Examination of DNA for quality/quantity (Yeild/Slot) - Analysis of DNA type (RFLP or PCR) - Interpretation

Used to find trace amounts of blood:

- Luminol
- Kastle-Meyers test

Used to find trace amounts of semen and saliva:

Semen: Prostate Specific Antigen aka PSA test.
Saliva: Amylase

Sources of DNA:

Teeth, blood, bone, saliva, hair, skin, feces, urine and semen.

PCR Inhibitors

Reduces efficiency of amplification of forensic samples. May inhibit polymerase enzyme or bind to DNA to prevent amplification.

Partial DNA profiles, false negatives.

Sources of Inhibition (PCR)

- Body fluids
- Substrate
- Reagents used in analysis

Blood PCR Inhibitor

Heme Immunoglobin G

Hair PCR Inhibitor

melanin

Vaginal/Buccal/Fecal PCR Inhibitor

Bacteria and microorganisms

Bone/Teeth PCR Inhibitor

Ca2+

Semen/Urine PCR Inhibitor

Semen: Polyamines
Urine: Urea

Substrate PCR Inhibitors

-Indigo dye
- Ca2+ in foods
- Tannic acid
- Organic compounds

Organic Extraction of DNA

DNA remains in aqueous phase while protein and other material move to organic layer. Preferred method.

What is used to lyse cells?

Detergent, Proteinase K, and DTT.

Sarkosyl

Used if lysis is done under refrigerated conditions. SDS precipitates out of solution at this temperature.

What is the detergent of choice for room temperature DNA extraction?

SDS (sodium dodecyl sulfate)

What does Proteinase K do? Why is it included?

Hydrolyzes histone proteins and inactivates nucleases. Aids in lysis of epithelial and WBC.
- Active in wide pH range (4-12.5)
- Active w/SDS present.
- Not affected by EDTA.

What is DTT?

- Reduces disulfides to dithiols.
- Allows for the release of DNA from protective proteins for further degradation of proteins by ProK.
- Essential for sperm cell lysis due to high disulfide content.

Removal of Denatured Proteins

- Uses PCl combo.
- Denatures proteins subsequently hydrolyzed w/ProK.
- Promotes separation of organic and aqueous phases.
- 25:24:1 ratio

What is used to purify and concentrate DNA?

Centrifugal filter units.

What is Chelex Extraction (PCR)?

An ion exchange resin that removes polyvalent metal ions that may break DNA down and inhibit PCR.

What does Chelex Extraction involve?

-Boiling sample in 5% suspension of DI water and Chelex 100.
- Suspension then centrifuged to separate resin and cellular debris from the supernatant containing denatured DNA.
-DNA can now be amplified.

When to not use Chelex?

When working with highly degraded DNA or low level DNA samples.

Advantages of Chelex

- Fast
- Simple
- Inexpensive
- Binds heavy metal cations
- Non-hazardous material
- Removes some PCR inhibitors

Disadvantages of Chelex

- Harsh extraction environment
- Degradation concerns
- Resin can inhibit PCR
- Isolated DNA is single-stranded
- Resin loses chelating after few hours
- Less effective on some sample types

What is FTA Paper?

Cellulose paper containing chemicals to lyse cells, denature proteins, protect DNA, and leave sample for identification w/o contamination.

Differential DNA Extraction

Used when processing sexual assault evidence. Based on differential lysis.

Qiagen DNA Mini Kit

Uses selective binding properties of a silica-based membrane to purify both nDNA and mtDNA. Buffer washes AW1 and AW2.

Methods of DNA Quantification:

- Yield Gels
- Spectrophotometry
- Fluorometry
- Slot Blot
- AluQuant
-qPCR (used in modern labs)

Yield Gels

Allows for estimation of concentration and quality of DNA in specimen. Uses electrophoresis in agarose gel matrix w/ fluorescent dye.

UV Spectroscopy

Only good for pure and concentrated DNA samples. Detects total amount of DNA.

Slot Blot Quantitation

- human specific
- 40bp probe
- DNA amount estimated by comparison of density of bands/bands observed to standards.

Traditional vs Real-Time PCR

Trad.: The amplified DNA product is detected in an end-point analysis.
Real-Time: The accumulation of amplification product is measured as the reaction progresses with product quantification after each cycle.

Traditional PCR Limits

- Endpoint detection is time consuming
- Results based on size
- End point changes
- Agarose resolution poor

Amelogenin

Marker used for sex determination. In DNA profile identification kits and amplify STR.

Electrophoresis

Phosphate groups on DNA give H+ ions, leaving fragments with neg. (-) charge. DNA molecules move away from cathode toward anode.

Agarose Gel

- Large pore size.
- Separates larger DNA molecules (RFLP, single PCR amp.).
- Good for single fragments and multiples 400bp or more.
- Low molecular mass DNA not well resolved.

Polyacrylamide Gel

- Small fragment separation is better.
- Essential for closely sized DNA molecule separation.
- multiplex PCR amplified STRs.
- Horizontal or vertical format.
- Can separate molecules that differ by a single nucleotide.

What are gel stains?

Intercalating agents that fluoresce under UV light.

Native vs Denaturing

Native: Separation of double stranded DNA.
Denaturing: Occurs in environment w/ single stranded DNA (Formamide or Urea)!!

DNA Separation Models

Ogsten: Small molecules through large pores.
Reptation: Large molecules through small pores.

What type of measurement is Electrophoresis?

RELATIVE not absolute. Must be compared to known standards.

Capillary Electrophoresis

- Effective tool for separation of compounds and materials.
- Used in medical and scientific comm..
- Used in forensic science for DNA. gunshot residue, explosive, drug, and pen ink analysis!!

Capillary Electrophoresis Process

- Buffer moves through capillary from power source.
- Osmotic and Endo-Osmotic flow.
- Osmotic flow is toward (+) anode.
- Source vial -> capillary -> detector -> destination vial.
- Fragments migrate toward anode resivoir.

Capillaries have viscous polymer solution.

Performance Optimized Primers

- Detect alleles differing in size by a single base.
Precision of less than 0.15 nucleotide s.d..
- Highly denaturing environment for DNA samples.