NAME: _____________________________________________ DATE: _______________ BLOCK: _____
DNA ISOLATION FROM BUCCAL CELLS
Overview: Analysis of DNA begins with extracting and isolating the
DNA from cells. DNA is used in genetic testing, forensic
investigations, and research labs. Human DNA is commonly extracted
from cheek cells, hair follicles, blood, and semen. DNA isolation
involves the following main steps: cell disruption and cell membrane
destruction, protein degradation, and precipitation of the DNA into
solution. There are many ways to accomplish each step.
This lab protocol uses a saline mouthwash to collect buccal (cheek)
cells, boiling to disrupt the cell membranes and lyse the cell, and
Chelex to collect and remove proteins. See the below for more
information about Chelex.
Chelex is a chelating resin (chemical structure as seen on the right); the
beads bind to or chelate the polar elements of the cell while DNA and
RNA stay in the solution. Chelex has a high affinity for heavy metals like
Mg2+, Ca2+, and Mn2+. Heavy metals can damage DNA and Mg2+ is a
cofactor of nucleases (enzymes that break down DNA), By binding to
these heavy metals, Chelex helps to minimize DNA damage. The
alkalinity of the solution and the high temperature break open the cells
and denature the proteins. DNA remains in the supernatant.
AMPLIFY DNA BY PCR
Use polymerase chain reaction (PCR) to make many copies of a short region of the TAS2R38 gene.
There are three steps to PCR. They are: denaturing, annealing, and elongating steps. The denaturing
steps separates complementary strands, the primers bond to the templates in the annealing step, and
finally the copies are made in the elongation step. Taq polymerase is used to elongate the strands.
Taq polymerase was first isolated from thermophilic bacterium Thermus aquaticus. These bacteria
thrive in extreme heat so their Taq polymerase can withstand the high temperatures required for PCR.
Contents
Ready-To-Go PCR Bead
Primer/loading dye mix
Your DNA sample
Purpose
Provides dNTPs (DNA building blocks), Taq DNA polymerase (adds
nucleotides to growing strands), reaction buffer (gives a stable reaction
environment), and MgCl2 (Mg2+ is an important DNA polymerization
cofactor)
Contains both a forward and reverse primer as well as a dye (so bands
can be seen gel electrophoresis)
Contains your entire genome; PCR will copy only a short region of the
TAS2R38 gene
DIGEST PCR PRODUCTS WITH HaeIII
The PCR products are digested by the HaeIII restriction enzyme. HaeIII has the following restriction
sites.
5’….GG˅CC….3’
3’….CC˅GG….5’
In the PTC lab and the TAS2R38 gene, the taster allele contains this restriction site and the nontaster
allele does not. The nontaster allele has a G instead of a C, which means a single base substitution
yields a restriction site in the taster allele and not the nontaster allele.
ANALYZE AMPLIFIED DNA BY GEL ELECTROPHORESIS
The loading dye was added in the PCR step along with the primers. The dye allows you to see the
bands because otherwise DNA would be invisible.
The plasmid pBR322 digested with the restriction enzyme BstNI is an inexpensive marker and produces
fragments that are useful as size markers in this experiment. The sizes of the DNA fragments in the
marker are: 1,857 base pairs (bp), 1,058 bp, 929 bp, 383 bp, and 121 bp. This marker or ladder can be
used as a guide to help estimate the sizes of your PCR fragments.
The buffer helps move electricity through the gel. Fragments move based on charge and size when
electricity moves through the gel. Since DNA has a negative charge, all fragments will move toward the
positive electrode in the chamber. Smaller fragments will move faster through the gel than longer
fragments.
CarolinaBLU and a final stain will help make the DNA visible so you can compare your bands.