DNA Extraction Techniques and Protocols

Chromosomes

Structures organizing DNA in the nucleus.

Base pairs

Units of DNA structure; 6 billion in chromosomes.

DNA typing

Identifying biological material donor.

STR profile

Short Tandem Repeat profile for DNA analysis.

DNA Isolation Goals

Lyse, separate, and Isolate

Lyse cells

Breaking cells to release DNA.

Isolate

Removal of proteins from DNA samples.

Nuclear DNA

DNA that is present in the nucleus of a cell and that is inherited from both parents

Nuclear DNA is present in all cells

false; not present in erythrocytes

A haploid cell has ________ Nuclear DNA

3 x 10^9 bp

All samples must be carefully handled regardless of the DNA extraction method to avoid:

- Sample-to-sample contamination

- Introduction of extraneous DNA

The extraction process is where the DNA sample is more susceptible to contamination in the laboratory than at any other time

true

MtDNA

Mitochondrial DNA, 16,000 base pairs long.

2-10 copies

A mitochondria has _____ mtdna copies and there are ______ mitochondria per cell

2-10, 10^3-10^4

MtDNA is especially useful in

hair shaft and bones

Haploid DNA

Single set of chromosomes, 3 billion bp, 3 picograms

Diploid DNA

Two sets of chromosomes, 6 billion bp, 6 picograms

How much DNA is required for DNA profiling (PCR)?

.5-1 ng

How many diploid cells are needed to obtain at least 1 ng (1000 pg) of DNA for a DNA profiling assay, given that the amount of nuclear DNA in a diploid cell is approximately 6.6 pg?

A) 100 cells

B) 150 cells

C) 200 cells

D) 250 cells

B) 150

RFLP requires 25-50ng

Restriction Fragment Length Polymorphism analysis.

DNA extracted from cell

if good extraction, M^R= 10^7, equivalent to ~ 20kb

Amount of DNA in Blood (leukocytes)

- 5 x 10^6 cells/ml

- range 30-60 µg/ml

Amount of DNA in semen

-50 x 10^6 cells/ml

(150 µg/ml)

- 5 x 10^6 leukocytes/ml in vasectomized individuals

The amount of DNA in saliva and tissues that are not blood or semen depends on

number of cells

chromosome is __ µm in length

5

DNA in chromosome is __ cm in length

5

DNA is compacted by

histones or protamines

All extraction strategies need to remove proteins

true

Factors Affecting Quantity and Quality of DNA

-Initial size of stain

- Environmental influences

- Extraction protocol

Environmental influences

- sunlight

- heat/moisture

• promotes growth of microorganisms

• deleterious effects of nucleases

environmental influences exacerbated by age of stain

true

Extraction protocol

shear forces--> nucleases --> normal extraction in efficiencies--> analytical prowess--> co extraction of inhibitors

DNA Extraction Methods

- Organic

- Chelex

- FTA

- Solid-phase extraction

Which type of DNA was effectively obtained using organic extractions?

High MW DNA

Organic extraction

Uses phenol/chloroform for DNA isolation.

Generic DNA Extraction Protocol

Solubilize cellular components

- denature/hydrolyze proteins

- remove denatured proteins

- clean the DNA (purification)

- quantitate the DNA (how much DNA?

Solubilization of Cellular Components

Buffer, EDTA, detergent, Heat

EDTA

chelating agent used to prevent Mg2+ ions required for nuclease activation, protecting it from degradation

Buffer

- maintain a stable pH level during the process so DNA doesn't denature

- help cells lyse & separate DNA from proteins

- produces pure, isolated DNA

SDS (detergent)

solubilizes cell, organelle membranes, and denatures protein to release DNA

Differential extraction

Isolating sperm DNA from mixed samples.

Proteinase K

Enzyme for digesting proteins during extraction to increase purity and yield of DNA.

*Added after buffer

Proteinase K acts on denatured proteins to produce

oligopeptides and amino acids

Phenol/chloroform

Deproteinizes dna

- Partition of solutes into the upper aqueous and lower organic phases

Where are denatured proteins found when using phenol in DNA extraction?

In phenol and at the interphase

Chaotropic agents

Disrupt hydrogen bonds, aiding protein denaturation.

- Ex: urea, guanidine

Chelex Extraction

An alternative and inexpensive procedure for DNA extraction.

What happens to magnesium ions during Chelex Extraction?

Mg ions are drawn in and bound by the resin which act as chelating group.

Issues with chelex extraction

-left dna single stranded

- too concentrated blood resulted in pcr inhibition

FTA Paper Extraction

a method for collecting and preserving DNA samples using filter paper cards

FTA paper extraction process

The cards are treated with chemicals that lyse cells and denature proteins, while protecting the DNA from degradation.

solid phase dna extraction

DNA is immobilized on a solid support, beads, or columns

Differential extraction process

1. remove portion of mixed stain

2. cell lysis buffer (sds, edta, and proteinase K)

3. centrifuge

4. remove supernatant (non-sperm fraction)

5. Another round of cell lysis buffer + DTT

6. sperm fraction

DTT

lyses sperm heads