Genetics-Lab-ACT-4 Notes

Genetics Laboratory
Activity 4
DNA EXTRACTION
DEOXYRIBONUCLEIC ACID (DNA)

❑ Considered the hereditary “code of life” because it possesses the information that
determines an organism’s characteristic and is transmitted from one generation
to the next
❑ Your DNA is your “genetic fingerprint”—this means that your DNA is like no one
else in the world!
❑ DNA is used every day by scientists and lawyers to help in criminal
investigations, paternity cases, cloning, etc.
DNA STRUCTURE
❑ DNA is a nucleic acid, made of the elements carbon, hydrogen, oxygen, nitrogen,
and phosphorous.
❑ Watson and Crick were the two scientists that were credited
with the discovery of its structure.
❑ DNA is made up of several nucleotides.
❑ Each nucleotide consists of a sugar, a phosphate and a
nitrogenous base.
❑ As nucleotide chains are formed, the DNA takes the shape
of a twisted ladder
❑ DNA is in the nucleus of almost every cell in your body.
❑ The length of DNA per cell is about 100,000 times as long as the
cell itself.
❑ However, DNA only takes up about 10% of the cell’s volume.
❑ This is because DNA is specially packaged through a series of events to fit easily
in the cell’s nucleus.
❑ The structure of DNA, the double helix, is wrapped around
proteins, folded back onto itself, and coiled into a compact
chromosome.
❑ Individual chromosomes can be studied using
microscopes, but the double helix of a chromosome is so
thin that it can only be detected through innovative, high-
tech procedures.
❑ Chromosomal DNA from a single cell is not visible to the
naked eye.
❑ However, when chromosomal DNA is from multiple cells,
the amassed quantity can easily be seen and looks like
strands of mucous-like, translucent cotton.
Buccal Cells Provide An Excellent Source of DNA
Extracting Human DNA

1. Buccal (cheek cells) can be harvested painlessly and in sufficient quantity to
visualize DNA extracted in a simple 4-step protocol
2. Collect cheek cells by swishing water in your mouth and using to gently scrape
cells off our cheeks. (The more vigorous and the longer that you swish, the more
cells are removed, and the more materials you’ll have from which to extract
DNA.)
3. Lyse the cell membranes by adding a detergent based cell lysis solution, which
allows the DNA to be freed (digestion). DNA is soluble in water, but much less
soluble in alcohol.
4. Alcohol will be slowly added, and DNA will precipitate to the solution, and you
will be able to see your own DNA!
5. As white, stringy material is thousands of DNA molecules stuck together.
• Pipette 3 ml water into the drinking cup
• Gently chew the inside of mouth for 30
seconds
• Gently – blood doesn’t help
• Take the water from tube into mouth and
move it around for 30 seconds
• Don’t swallow the water
• Carefully spit the water back into the cup
• Add 2 ml of lysis buffer to the test tube for

DNA extraction
• Pour the contents of the cup into the test tube
• Put the cap on the tube
• Gently swirl the tube to mix
• Shaking cuts, the DNA leading to short strands
at the end of the experiment
Lysis Buffer
❑ For extraction of DNA the lysis buffer will commonly contain sodium dodecyl
sulfate (SDS) and sodium hydroxide (NaOH)
❑ Lysis buffers break the cell membrane by changing the pH.
❑ SDS solubilizes the cell membrane.
❑ NaOH helps to break down the membrane, and disrupts the hydrogen bonding
between the DNA bases, converting the double-stranded DNA to single-stranded
DNA.
Importance of Cell Lysis Buffer

❑ It lyses the nuclear membrane as well as a cell membrane.
❑ It maintains the pH during the DNA extraction.
❑ Lysis buffer maintains the integrity of the DNA (protect DNA from lysis)
❑ It separates DNA from other cell debris.
❑ It protects DNA from acidic degradation.
❑ General chemicals used in lysis buffer are Tris, EDTA, SDS, CTAB, Triton X100,
MgCl2, KCl, NaCl and other detergents.
• Add 0.25 ml of Proteinase K solution to the tube
• Put the cap on the tube
• Gently swirl the tube to mix
• Place the tube in the 56oC water bath for 10 minutes
• Add 0.5 ml of 0.5 M NaCl to the tube and swirl the

tube gently to mix
• Hold the tube at 45o and carefully pour in 10 ml of cold
isopropyl alcohol
• Leave the tube on the desk for 5 minutes
• It is very important not to shake the tube
• After 5 minutes DNA should have precipitated at the
interface between the lysis buffer and the alcohol
• Do not shake or invert the tube
DNA Precipitation
❑ Adding salt helps to neutralize the DNA charge
and make the molecule less hydrophilic, meaning
it becomes less soluble in water.
❑ The salt also helps to remove proteins that are
bound to the DNA and to keep the proteins
dissolved in the water.
❑ The high concentration of salt causes the
proteins to fall out of solution, and then
centrifugation separates the soluble nucleic acid
from the cell debris and precipitated protein
What is the purpose of DNA extraction?
❑ The ability to extract DNA is of primary importance to studying the genetic
causes of disease and for the development of diagnostics and drugs.
❑ It is also essential for carrying out forensic science, sequencing genomes,
detecting bacteria and viruses in the environment and for determining paternity.
Laboratory Activity 4 – DNA Extraction

• Schematic Diagram of the DNA Extraction from Human Cheek Cells.
• Answer the guide questions.

Genetics-Lab-ACT-4 Notes

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Genetics Laboratory

DEOXYRIBONUCLEIC ACID (DNA)

Extracting Human DNA

• Add 2 ml of lysis buffer to the test tube for

Importance of Cell Lysis Buffer

• Add 0.5 ml of 0.5 M NaCl to the tube and swirl the

Laboratory Activity 4 – DNA Extraction

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