Unit 2 Flashcards
Leading Strand
Continuous synthesis continues in a 5 to 3 direction
Lagging Strand
Discontinuous synthesis produces 5’ to 3’ DNA segments
Lagging Strand Segments
Okazaki Fragments
Significance of Primer
To tell the polymerase where to bind on the old strand of DNA to build the new strand
DNA Polymerase Read VS Build
3’ to 5’ VS 5’ to 3’
PCR Technology Was Developed By
Mullis who won the Nobel Prize in 1993 for it
Steps of PCR
Denaturing, annealing, extending
Denaturing
Breakdown of DNA
Separate the DNA into single strands by heating it to 94-98 degrees Celsius for one to two minutes, and the heat breaks the hydrogen bonds
Annealing
Lower the temperature back down to 50-65 degrees Celsius in order to get the primers to anneal, or base pair, to their complementary sequences
Extending
Raise the temperature back up to 72 degrees Celsius which allows the DNA polymerase (which works to copy the DNA) to attach at the primer site and copy the target section of DNA
Primer Dimers Tell You That
PCR worked
In Vitro
Outside the body
In Vivo
Inside the body
DNA Replication Comparison
Two copies, uses helicase, in vivo, uses helicase, primase, and ligase, has okazaki fragments
PCR Comparison
Millions of copies, uses heat, in vitro, consistent, doesn’t use main enzymes
DNA Replication and PCR Same
Make copies of DNA, use polymerase
Most Common Type of Sequencing
Sequencing by Synthesis (SBS)
Next Generation Sequencing
Faster, easier, cheaper, and more accurate
DNA Barcoding in Animals
Looks at COI gene (cytochrome oxidase)
658 bp mitochondrial DNA (mtDNA)
Field of Bioinformatics
Application of tools of computation and analysis to the capture and interpretation of biological data
International Barcode of Life Project
Mega project aimed at identifying and categorizing all life on Earth
Illuminate biodiversity to save our living planet
IBoLP Relate to Estimated Number of Identified Species Compared to Yearly Extinction Rate
As species diversity increases, the likelihood of extinction decreases
Scientists believe that species are going extinct at a faster rate than we can identify them with classic taxonomy
Classic Taxonomy VS DNA Barcoding
Convergent evolution makes identification difficult
Same species can look very different
Different species can look similar
Classifying organisms requires advanced training
Time consuming, slow process
Species are going extinct faster rate than can be identified
Huge number of species
Threats to outpace the rate of species discovery
Some situations it is impossible to identify using classic taxonomy
DNA barcoding uses little DNA to identify and compare an organism
DNA Barcoding Contraversy
Concerns of accuracy
Scientists disagree that regions sequenced are not best for species identification
DNA barcoding minimizes the value of taxonomic identification
Uses of DNA Barcoding
Identify species, biodiversity, mislabeling, interactions, habitat requirements, migration patterns
Understand history of an ecosystem
Detect disease, illegal products or species prescence through environmental DNA (eDNA)
Stop wildlife trafficking, poaching, and illegal trade
What Can DNA Barcoding Not Answer
Identifying hybrid species, single gene regions, or closely related species
DNA Regions Amplified in Plants, Animals, and Fungi
Plants rbcL, chloroplast, MatK gene
Animals CO1, mitochondrial gene
Fungi ITS, nuclear intron near ribosomal RNA gene
Conserved but variable
Highly Conserved But Variable
A segment of DNA that remains similar across different species but also polymorphic
Purpose of Barrier Filter Tips on the Micropipettes
Avoids cross-contamination, keeps aerosol out
Why Use a Very Small Piece of DNA To Perfrom Extraction On
Large samples introduce cellular components and debris that can inhibit PCR
Different Buffers Usage
Resuspension provides optimal pH and ideal condition
Lysis breaks down cell membranes and releases cellular components
Neutralization neutralizes lysate and digests RNA present
Wash removes excess and unbound components
What Was Added to PCR Tube for PCR
Master mix contains DNA nucleotides and polymerase
Corresponding primer for specific type of organism
Primer and DNA were added seperately
Electropherogram
Visual representation of your sequence data where each nucleotide is assigned a different color by the computer software
QS Score
Average of all of the Phred scores for your sequence data
≥ 0 is excellent
≥ 50 is issues with individual nucleotide bases
≥ 40 is enough issues with bases that data may not be accurate
Phred Score
Quality score for each individual nucleotide
Accuracy of the nucleotide call
20 is the cut-off for a high-quality sequence
Peak Quality
Clear, high peaks with little to no background noise mean high quality Background noise refers to many overlapping peaks in same location
BLAST
Quickly identify any close matches to your sequence in sequence databases
MUSCLE
Align your sequences of DNA Barcodes to visually represent data between two or more sequences
Lines and Grey Areas in a DNA Barcode
Both sequences are the same, it is a gray bar
Dashes mean a place where sequences didn’t align, or one sequence has more nucleotides in that spot than the other
Sanger Sequencing/Chain Termination
Fredrick Sanger
Sequences small sections of DNA
Replicates fragments of DNA using PCR
Shotgun Sequencing
Sequences entire genome
Genome is cut into smaller fragments using restriction enzymes.
These fragments are sequenced using Sanger sequencing
A computer is used to overlap the fragments to piece together original sequence
Used for first entire human genome in Human Genome Project
Sequencing by Synthesis/SBS
Most common
Nucleotides are labeled with fluorescent tag
DNA fragment is sequenced as PCR occurs
Each nucleotide is incorporated into growing strand, fluorescent tag is removed and emits a light flash that the computer reads
Nanopore Sequencing
One side of DNA through a nanopore in membrane
Charged ions pulls DNA through the pore in the synthetic membrane one nucleotide at a time
Base is identified by measuring differences in their effect on ions
High error rates but little power needed
Enzymes Involved in DNA Replication
Helicase, binding proteins, primase, DNA polymerase III and I, and ligase
Helicase
Unzips DNA
DNA Polymerase
III performs elongation in eukaryotes and creates a sugar-phosphate bond between the nucleotides
I is enzyme that removes RNA Primers and adds nucleotides to section
Steps of DNA Replication
- Helicase binds to orgin and seperates strands
- Binding proteins keeps strands apart
- Primase makes short stretch of RNA on the DNA template
- DNA polymerase adds DNA nucleotides to RNA primer
- DNA polymerase proofreads and checks for replaces incorrect bases
- Enzymes remove RNA primers and ligase seals sugar-phosphate backbone
Reading DNA
Basis of orientation of sugar phosphate backbone
5′ end has free phosphate group whereas 3′ end has free hydroxyl group
When Does DNA Replication Happen Naturally
Prior to cell division during the synthesis phase of the cell cycle
After 20-30 Cycles How Many Copies of the Original DNA Sequence Has Been Made
1 million
Primer Dimer
A small band that runs ahead of the other bands
Formed when primers bond to each other and replicate themselves
Conversions
1uL = a millionth L
1 mL = 1000 uL
1 L = 1000 mL
1L = 1,000,000 uL
Why is DNA Temperature Sensitive
Buffers have different jobs adn DNA needs to be released without being denatured
DNA Throughout the Extraction Process
Wash buffer bound to beads washed out
DI water new tube released dna from beads
Spin column sometimes above beads sometimes in beeds
Role of Supernatant and Matrix
Supernatant DNA
Matrix DNA was bound to matrix so DNA wouldn’t get washed through
Sequencing Used and Why
Sagar sequencing to sequence one gene
Shotgun is using sangar but every gene
Nanopore too expenxive
Synthesis special equipment
CRL Score
How long the computer could read the sequence in base pairs
Role of Primers and Primase in DNA Replication
Tells polymerase where to go primase places primer
Primer Dimer But No Other Bands Meaning
PCR worked but they didn’t extract DNA
What’s in Sample of DNA to Replicate Via PCR
DNA sample, polymerase, DNA nucleotides, primer
