DNA Extraction

What are the goals of DNA Extraction?

Lyse cells and separate DNA from other cellular components in a way that's compatible to downsteam assays

All samples of are handled carefully regardless of the extraction method to avoid

contamination, introduction of extra DNA (the extraction process is where the DNA sample is most susceptible to contamination

Where is an organism’s DNA found?

nucleus and mitochondria

Where is the majority of an organism’s DNA found

nucleus

The nucleus contains how many copies of an organism’s DNA

two

A cell’s chromosomes contain how many base pairs of DNA

6 billion

How many base pairs of nuclear DNA are in a haploid cell?

3 billion (3×10^9)

How much DNA is contained in the mitochondria?

1600 bp with 2-10 copies each

What are the amount of mitochondria in the cell?

10³ - 10^4

MtDNA is especially useful where

in hair shaft and bone

Why is mitochondrial DNA used less often than nuclear DNA in forensic testing?

There's much more mitochondrial DNA in a cell, which makes the testing more sensitive and harder to interpret

How many picograms of DNA is in a diploid cell?

6 picograms

How much DNA do analysts need for CURRENT dna analysis?

0.5-1.5 ng

How would you convert from nanograms to cells?

(Nanograms * 1000)/6

How much dna/ml is there in blood?

30 micrograms/ml

How much dna/ml is there in semen?

150 micrograms/ml

How many cells/ml is there in a vasectomized individual's semen?

5*10^6 cells/mL

How many cells/ml is there in a normal individual's sperm?

50*10^6 cells/mL

What are some environmental factors that can affect the quality of the sample?

Size of stain, sunlight, heat, moisture

What are some protocol factors that can affect the quality of the sample

Shear forces can result in ripping of dna, analysts ability to properly separate DNA with pipette

What are the different types of extraction methods?

organic, chelex, FTA, solid-phase

WHat type of DNA extraction results in the highest molecule weight of DNA extracted?

Organic extraction

What type of extraction is most ocmmonly used in today's labs?

solid-phase extraction

What are the generic steps of DNA extraction?

1. Solubilize cellular components 2. Denature/hydrolyze proteins 3. Remove/denature proteins 4. Clean DNA 5. Quantitate

What are the types of solubilization used in organic extraction

EDTA or SDS (detergent)

What is the purpose fo EDTA in extraction?

Chelatess the Mg2+ required for nucleases to work. Also prevents aggrregration of nucleic acids

What is the purpose of SDS detergent in extraction

Solubilized cell and organelle membranes

In the past, how was solubilization achieved?

Sample was heated at 56 C overnight. Resulted in denaturing of DNA

What is the purpose of Proteinase K in extraction?

Is able to free DNA from histones

Under what circumstances is Proteinase K active?

Active in the presence of detergent. Specific for aromatic and hydrophobic proteins

Explain the use of phenol in extraction

Deporteinization. CHCl3 improves efficeincy and Isoamyl alcohol prevents foaming

What is the purpose of chaotropic reagents in extraction?

Disrupt hydrogen bonds. Examples include urea and guanidine

What the the purpose of phenolchloro in extraction?

Separates phases. Proteins are located in organic and interphase while all DNA is in aqueous phase

How is ethanol and isopropyl alcohol used in extraction?

Ethanol competess for DNA binding with water, resulting in the formation of a pellet

How does chelex extraction differ from organic extraction.

Utilized an ion-exchange resin that takes on the role of EDTA in capturing and removing Mg2+. Required the boiling of DNA samples resulting in single-stranded products. Most blood samples would have too much heme and require further extraction.

How does FTA paper extraction work?

Blood is dropped onto FTA paper where hte cell is lysed and DNA from white blood cells are trapped in paper's matrix. A punch of the paper is removed and heme is removed in purification process

How does solid-phase extraction work?

DNA is bound to silica particles while everything else is washed away

How does magnetic bead extraction work?

DnA is bound to magnetic beads at pH< 7.5. A magnetic stand makes the beads stick to the side of the tube while impurities remain in solution

What is the goal of differential extractions?

Separates sperm from non-sperm cells

How does differential extraction work?

Sperm cells have a different morphology than other cells, including a tough exterior. Nonsperm cells are targeted first so only sperm remainds.