FS531L: Advanced Forensic Biology Lab Quiz 1 Study Terms

What do we need to be careful about when collecting biological samples?

1) contamination

2) degradation of DNA (We want to slow or stop degradation)

Define contamination.

- addition of a different person's physiological material after the crime or during collection

- may be inadvertent (accidental)

What happens when contamination occurs?

invalidates DNA evidence completely (changing genotype)

What are some important precautions to take to avoid contamination?

- Always wear gloves/face mask

- Change gloves between samples (can double glove to make this easier)

- Use clean instruments

- Proper labeling and packaging of samples is a must

- Avoid contaminating areas where DNA might be present by not touching it with bare hands, sneezing, or coughing on the site.

- Use clean disposable gloves, changing them between items.

- Each item of evidence should be packaged separately

To avoid contamination, proper attention should be paid to...

- collection of the sample

- preservation, handling, and storage of sample

What are a few ways to slow degradation of DNA?

- Removing moisture

- Lowering temperature

What are the overall steps in DNA analysis?

1) Extraction

2) Quantitation

3) Amplification

4) Separation

5) Data Analysis & Interpretation

What is a DNA extraction?

separates DNA from other cellular components; in the cell, proteins help package and protect DNA, but they inhibit DNA analysis

What are the steps involved in DNA extraction?

1) Cell and tissue disruption

2) Lysis of organelles and membranes

3) Removal of proteins and other cellular components

What are the different types of extraction?

1) Organic

2) Differential

3) Paramagnetic (silica or dextran)

-Silica: Promega DNA IQ, Qiagen's QiaAmp, Automated systems

-Dextran: Life Technologie's PrepFiler

4) Chelex

What is the difference between DNA extraction and isolation?

Extraction: get biological material off of what it is stuck to

Isolation:

- separates DNA from other cellular components

- in the cell, proteins package and protect DNA but inhibit DNA analysis

Need to:

1) Disrupt cells and tissues

2) Lyse organelles and membranes

3) Remove proteins and other cellular components

What are the overall steps of the organic extraction?

1) Add SDS and proteinase K and digest buffer

2) Incubate with buffer

3) Add phenol:chloroform:isoamyl alcohol and centrifuge

4) Transfer aqueous phase and concentrate via precipitation or centrifugal filter

* also includes EtOH washes*

What is present in the digest buffer of the organic extraction? What do each of them contribute?

- Tris Buffer

=> help maintain a stable pH for extraction; creates ideal environment for extraction

- NaCl

=> introduces more positive charge (DNA is negatively charged)

- SDS

=> denatures proteins and dissolves membranes (cell lysis); destroys organelles, lipids, etc.

- EDTA

=> chelating agent; binds metal ions, like Mg, prevents DNA degrading enzymes (such as DNase) from working since they need metal ions as cofactors.

What is the function of Proteinase K in the organic extraction?

destroys proteins (frees DNA from proteins, like histones) by cleaving peptide bonds

What does Phenol/Chloroform/IAA do in the organic extraction?

separates into organic and aqueous layers, DNA is found in the aqueous

Why are washes performed in the organic extraction?

to remove interferences

What is TE used for in the organic extraction?

to help concentrate DNA

What are the pros and cons of the organic extraction?

Pros:

- thorough and efficient

- recovers double-stranded DNA

Cons:

- time-consuming/lengthy

- uses hazardous chemicals

- requires a lot of sample transfer, which introduces possibility of error and contamination

What is the organic differential extraction? Explain the general process.

separates epithelial and sperm fraction

1) Add SDS and proteinase K (and buffer)

2) Incubate and centrifuge

3) Transfer epithelial fraction (aqueous cell lysate) and wash spermatozoa pellet

Epithelial Fraction

4) add P:C:IAA and centrifuge

5) Transfer aqueous and concentrate

Sperm Fraction:

4) add SDS, proteinase K, and DTT

==> spermatozoa fraction

5) Add P:C:IAA and centrifuge

6) Transfer aqueous and concentrate

How can male profile get into the epithelial fraction in differential extractions?

degraded samples, sperm will lyse more easily

How can female profile get into the sperm fraction?

if there is a high abundance of epithelial cells compared to sperm cells

List the steps of Promega's DNA IQ Extraction.

Paramagnetic (silica)

1) Add Lysis Buffer

2) Add Magnetic Beads and Wash sample

3) Elute DNA

How are chaotropic salts used in the Promega DNA IQ extraction.

destroy protein components and allow DNA/RNA to stick to beads.

What are the pros and cons of the Promega DNA IQ extraction?

Pros:

- fast

- skips use of hazardous chemicals

- everything stays in the same tube (reduces risk of error and contamination)

Cons:

- cannot be used with tissue samples

- resin can only hold so much DNA

Briefly list the steps of the Qiagen QiaAmp DNA Investigator Kit.

1) Sample Addition

2) Lysis

3) Bind DNA (to silica beads)

4) Wash

5) Elute

==> ready-to-use DNA

Explain how automated DNA extraction robots operate.

1) Sample

2) Magnetic particles added to sample

3) Magnetic separation

4) Wash

5) Magnetic separation

6) Elute

==> pure, high-quality DNA

What is the ABI PrepFiler Extraction? List basic steps.

paramagnetic extraction with dextran beads.

1) Lysis

2) Substrate Removal

3) Bind

4) Wash

5) Elute

6) Purified DNA

What are the pros and cons of the PrepFiler Extraction?

Pros:

- greater binding capacity (compared to DNA IQ)

- fast

- eliminates use of harmful compounds

- everything stays in the same tube (reduces risk of error/contamination)

Cons:

- cannot be done on tissue samples

- resin can only hold so much DNA

What is the only instance Chelex extractions are used for today?

reference samples

Explain the general steps of the Chelex extraction.

1) Add water

2) Add Chelex resin

3) Incubate and centrifuge

Chelating Resin and Buffer

4) Heat for 20 mins

5) Spin

6) Remove supernatant and add to new tube

DONE

What is the Chelex resin?

styrene divinylbenzene copolymers that bind polyvalent metal ions such as magnesium (preventing degradation of DNA)

How is DNA released in the Chelex extraction?

heat

What are the pros and cons of the Chelex extraction?

Pros:

- cheap

- no hazardous chemicals

- good when in need of immediate results

- can be easily automated

- uses only 1 tube for extraction

Cons:

- resin is PCR inhibitor

- no wash step to clean sample

- heating step creates single-stranded DNA

What are examples of ways that we test for/prevent contamination concerns?

- Must extract knowns and unknowns separately to avoid possibility of cross-contamination

- Use reagent controls to screen the extraction process for possible points of contamination

=> blank tube that undergoes same samples process as samples

=> test reagents for contamination

Extraction control:

- demonstrates successful extraction techniquw