Wolbachia Introduction
Since Wolbachia can not be cultured on a Petri dish, PCR is the best option. In order to determine
if insects are infected with the bacteria Wolbachia, genomic DNA must be obtained. The DNA
extracted will not only be that of the insect, but also the bacteria if present.
DNA EXTRACTION & PURIFICATION - Total genomic DNA can be isolated from tissues using a
variety of protocols but all involve two steps: cell lysis (breaking cell & nuclear membranes) and
DNA purification (separating DNA from the rest of the cellular debris).
Heat is used to rupture membranes and free genomic DNA. Enyzmes in the cytoplasm now have the
opportunity to damage the DNA so a chelating agent (Instagene Matrix) is used to bind the
cofactor (magnesium ion) of DNases. DNA will be less dense that other components in the cellular
debris so centrifugation will separate the heavier components and InstaGene (IG) from the DNA.
AVOID the pellet when obtaining purified DNA in the SUPERNATANT! IG will inhibit your PCR
reaction!
DNA AMPLIFICATION - In order for DNA to be analyzed on a gel, the sequence of interest
(amplicon) must be amplified a billion times in a thermal cycler. WE are not amplifying the entire
genome! To identify the target sequence requires primers. The building blocks and chemicals needed
for synthesizing the amplicon are found in the PCR bead.
PCR is the process developed by Kary Mullis that will double the amount of amplicon during each
cycle. The three steps: 1) Denature -highest temperature causes helix to open, 2) Anneal – coolest
temperature; primers attach to target site, and 3) Extension – Taq Polymerase attaches
complementary bases. To produce a billion copies of the amplicon requires at least 30 cycles.
1-Aliquot
2-Allele
3-Amplicon
4-Anneal
5-Biotechnology
6-Chelate
7-Cofactors
8-Denature
9-DNases
10-dNTPs
11-Ethidium
bromide
12-Eukaryotes
13-Exon
Summer 2013
The division of a quantity of material into smaller, equal parts
A variation of a gene on a particular chromosome
Target sequence of DNA that will be copied many times
The coolest step of PCR; the binding of primers to complementary sequences on the
template DNA strands
The manipulation of an organisms (microbes, plants or animals) DNA to help solve
human problems
To bind metal ions in solution; a common chelating agent is EDTA (ethylenediamine
tetraacetic acid)
Ions or small molecules needed by an enzyme to function properly; Example: Taq
DNA polymerase needs Mg+2 in order to function properly.
The first step of PCR; high temperatures cause DNA strands to separate
Digestive enzymes that degrade DNA
Deoxynucleotide triphosphates (dATP, dTTP, dGTP, dCTP); used in synthesizing
DNA
A fluorescent dye molecule that intercalates between DNA base pairs and fluoresces
when exposed to UV
Organisms that are made up of cells containing a membrane-bound nucleus that
contains DNA
The region of a transcribed mRNA molecule that gets spliced together and leaves the
nucleus for translation into protein sequence
K. McKone
14-Extension
15-Genome
16-InstaGene
Matrix
17-Intron
18-Lysis
19-Master Mix
20Microcentrifuge
21-Molecular
Biology
22-Nucleotides
23-PCR
The last step of PCR; process whereby Taq polymerase adds dNTPs on the ends of
the primers. Extension follows the base pairing ules and proceeds in the 5’ to 3’
direction.
The complete set of genetic instructions in the cells of a particular type of organism
Microscopic beads that bind divalent cations in solution; the binding of divalent
cations prevents their availability to enzymes that can degrade the DNA template
The region of a transcribed mRNA that is spliced out of mRNA and is not translated
into protein sequence
The process of rupturing a cell to release its constituents (especially genomic DNA)
A solution for the PCR reaction; usually contains the forward and reverse primers
Biotech instrument used to separate substances
The study of genes and the molecular details that regulate the flow of genetic
information from DNA to RNA to proteins
The monomer of DNA or RNA; consists of a sugar (deoxyribose or ribose),
phosphate, and nitrogenous base (adenine, thymine, cytosine, or guanine, with uracil
in place of thymine in RNA)
Polymerase Chain Reaction; the process of amplifying or synthesizing DNA within
a test tube
24-PCR Ready
Beads
Tubes containing a white pellet composed of dNTPs, buffer, magnesium, and Taq
polymerase
25-Primers
Oligonucleotides; A short sequence of nucleotides (usually 16-24 bases in length)
that recognizes a particular sequence of DNA; primers are usually synthesized in a
lab
26-Reagents
Usually solutions or mixtures of various solutions needed to conduct an experiment
27-Restriction
Enzymes
Enzymes produced by bacteria for protection against viral DNA
28-Taq DNA
polymerase
Thermostable DNA polymerase that was isolated from the thermophilic bacterium
Thermus aquaticus; this DNA polymerase is commonly used in PCR reactions
29-Template
The strand of DNA that contains the target sequence that will be copied into its
complementary strand
30-Vortex
Biotech machine used to mix substances
Summer 2013
K. McKone