For Bio midterm

Alphonse Bertillon

The Father of Anthropometry (Bertillonage).

Dr. Edmund Locard

Created the Locard Exchange Principle.

Locard Exchange Principle states that:

Whenever two objects come into contact with each other, traces of each are exchanged.

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. Aka 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)

PCR (picture)

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

DNA Replication (picture)

PCR primers consist of:

Two oligonucleotides that 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

Flowchart for Organic Extraction of DNA

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.