Summary BIO 1414 Lab Review 1 Study Guide

LAB PRACTICAL #1 REVIEW

MOLECULAR PHYLOGENY

Learn the process and utility of DNA barcoding
- Barcodes distinguish between different species with one group because they show the
variation and difference in each DNA sequence
- During the process of DNA barcoding, discrete gene loci are chosen as barcode regions,
then unique DNA primer sets are determined that used to amplify these same regions
across a range of species by the polymerase chain reaction (PCR), the result will be
sequenced and compared
- Several barcoding primers:
o GMOs: The primers amplify a 190 base pair fragment of the EPSPS gene
o Fungi: The primers amplify a 560 base pair fragment that is an
internal transcribed spacer that surrounds the 5.8S ribosomal RNA gene
o Plants: The primers amplify a 600 base pair fragment of the chloroplast gene rbcL
o Animals: The primers amplify a 700 base pair fragment of the mitochondrial
cytochrome c oxidase subunit 1 gene
o Prokaryotes: The primers amplify a 1500 base pair fragment that is within the 16S
rDNA, that encodes for the 16S ribosomal RNA
Steps to PCR
- Denaturation - 94-98°C, separates the template DNA into single strands
- Annealing - 55-70°C, allows primers to bind to the template with their specific
complementary sequences
- Extension - 65-72°C, allows the DNA polymerase to build complementary
DNA extending from each primer 3'OH
Analysis of barcoding PCR products
- The size of the fragments produced from the PCR reaction will be determined
by comparing their migration to the migration of a marker, which is also run on the gel
and has fragments of a known size
- When plotting the log graph for analysis, treat it like a normal graph
o Include graph title, x-axis, and y-axis with units and a legend for
when there are multiple lines/bars
- X-axis should be the distance traveled (cm) and y-axis should be the
size of the DNA fragment/marker (bp)
- The distance of a fragment of DNA moves into the gel is inversely
proportional to the size of the fragment in bp
o Which means a smaller sized fragment would more likely to
travel further
Learn pros and cons of how to detect genetically modified foods
- Arguments in favor GMOs:
o Agricultural: Increased yield, and the ability to feed an ever-growing population

, o Environmental: Reduced use of pesticides, herbicides, and fuel
o Nutritional: Improved quality of food
o Disease prevention: Foods that work like edible vaccines
- Arguments against GMOs:
o Exposure to possible allergens and toxins
o Harm to the environment if not contained
o Antibiotic resistance
o The spread of introduced genes to non-target plants by outcrossing and pollen
drift
How many primers does it take to amplify a single PCR product?
- It takes 2 different primers to amplify a single PCR product because one primer for
each end of the sequences is needed (3' and 5')




Learn the types of DNA modification
- Deletion: Occurs if the detached fragment of DNA does not reattach to the original DNA
sequence
o Becomes obvious when then the lengths of the two DNA sequences are different
(one is shorter than the other)
o Example: Species A has a DNA sequence of ATGCGTTCGA and species B has a
DNA sequence of ATGCCGA. Here, the sequence “GTT" is being deleted
- Duplication: Occurs if the detached fragment of DNA is duplicated and both copies end
up in the DNA sequence
o Becomes obvious when the lengths of the DNA sequences are different
o May produces more bases
o Example: Species A has a DNA sequence of CTGGATATG and species B has a
DNA sequence of CTGGATGATATG. Here the sequence of “GAT" is being
duplicated and repeats twice in the DNA sequence
- Inversion: Occurs if the detached fragment of DNA reverses its orientation and attaches
back to the DNA sequence
o No obvious length difference detected
o Example: Species A has a DNA sequence of AGGCATTCA and species B has a
DNA sequence of AGGTACTCA. Here, the sequence "CAT" (starting from the
4th base) is reversed and attaches back to the original sequence