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Biotechnology > Unit 2 > Flashcards

Unit 2 Flashcards

1
Q

Leading Strand

A

Continuous synthesis continues in a 5 to 3 direction

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2
Q

Lagging Strand

A

Discontinuous synthesis produces 5’ to 3’ DNA segments

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3
Q

Lagging Strand Segments

A

Okazaki Fragments

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4
Q

Significance of Primer

A

To tell the polymerase where to bind on the old strand of DNA to build the new strand

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5
Q

DNA Polymerase Read VS Build

A

3’ to 5’ VS 5’ to 3’

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6
Q

PCR Technology Was Developed By

A

Mullis who won the Nobel Prize in 1993 for it

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7
Q

Steps of PCR

A

Denaturing, annealing, extending

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8
Q

Denaturing

A

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

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9
Q

Annealing

A

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

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10
Q

Extending

A

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

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11
Q

Primer Dimers Tell You That

A

PCR worked

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12
Q

In Vitro

A

Outside the body

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13
Q

In Vivo

A

Inside the body

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14
Q

DNA Replication Comparison

A

Two copies, uses helicase, in vivo, uses helicase, primase, and ligase, has okazaki fragments

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15
Q

PCR Comparison

A

Millions of copies, uses heat, in vitro, consistent, doesn’t use main enzymes

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16
Q

DNA Replication and PCR Same

A

Make copies of DNA, use polymerase

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17
Q

Most Common Type of Sequencing

A

Sequencing by Synthesis (SBS)

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18
Q

Next Generation Sequencing

A

Faster, easier, cheaper, and more accurate

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19
Q

DNA Barcoding in Animals

A

Looks at COI gene (cytochrome oxidase)
658 bp mitochondrial DNA (mtDNA)

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20
Q

Field of Bioinformatics

A

Application of tools of computation and analysis to the capture and interpretation of biological data

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21
Q

International Barcode of Life Project

A

Mega project aimed at identifying and categorizing all life on Earth
Illuminate biodiversity to save our living planet

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22
Q

IBoLP Relate to Estimated Number of Identified Species Compared to Yearly Extinction Rate

A

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

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23
Q

Classic Taxonomy VS DNA Barcoding

A

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

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24
Q

DNA Barcoding Contraversy

A

Concerns of accuracy
Scientists disagree that regions sequenced are not best for species identification
DNA barcoding minimizes the value of taxonomic identification

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25
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
26
What Can DNA Barcoding Not Answer
Identifying hybrid species, single gene regions, or closely related species
27
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
28
Highly Conserved But Variable
A segment of DNA that remains similar across different species but also polymorphic
29
Purpose of Barrier Filter Tips on the Micropipettes
Avoids cross-contamination, keeps aerosol out
30
Why Use a Very Small Piece of DNA To Perfrom Extraction On
Large samples introduce cellular components and debris that can inhibit PCR
31
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
32
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
33
Electropherogram
Visual representation of your sequence data where each nucleotide is assigned a different color by the computer software
34
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
35
Phred Score
Quality score for each individual nucleotide Accuracy of the nucleotide call 20 is the cut-off for a high-quality sequence
36
Peak Quality
Clear, high peaks with little to no background noise mean high quality Background noise refers to many overlapping peaks in same location
37
BLAST
Quickly identify any close matches to your sequence in sequence databases
38
MUSCLE
Align your sequences of DNA Barcodes to visually represent data between two or more sequences
39
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
40
Sanger Sequencing/Chain Termination
Fredrick Sanger Sequences small sections of DNA Replicates fragments of DNA using PCR
41
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
42
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
43
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
44
Enzymes Involved in DNA Replication
Helicase, binding proteins, primase, DNA polymerase III and I, and ligase
45
Helicase
Unzips DNA
46
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
47
Steps of DNA Replication
1. Helicase binds to orgin and seperates strands 2. Binding proteins keeps strands apart 3. Primase makes short stretch of RNA on the DNA template 4. DNA polymerase adds DNA nucleotides to RNA primer 5. DNA polymerase proofreads and checks for replaces incorrect bases 6. Enzymes remove RNA primers and ligase seals sugar-phosphate backbone
48
Reading DNA
Basis of orientation of sugar phosphate backbone 5′ end has free phosphate group whereas 3′ end has free hydroxyl group
49
When Does DNA Replication Happen Naturally
Prior to cell division during the synthesis phase of the cell cycle
50
After 20-30 Cycles How Many Copies of the Original DNA Sequence Has Been Made
1 million
51
Primer Dimer
A small band that runs ahead of the other bands Formed when primers bond to each other and replicate themselves
52
Conversions
1uL = a millionth L 1 mL = 1000 uL 1 L = 1000 mL 1L = 1,000,000 uL
53
Why is DNA Temperature Sensitive
Buffers have different jobs adn DNA needs to be released without being denatured
54
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
55
Role of Supernatant and Matrix
Supernatant DNA Matrix DNA was bound to matrix so DNA wouldn't get washed through
56
Sequencing Used and Why
Sagar sequencing to sequence one gene Shotgun is using sangar but every gene Nanopore too expenxive Synthesis special equipment
57
CRL Score
How long the computer could read the sequence in base pairs
58
Role of Primers and Primase in DNA Replication
Tells polymerase where to go primase places primer
59
Primer Dimer But No Other Bands Meaning
PCR worked but they didn't extract DNA
60
What's in Sample of DNA to Replicate Via PCR
DNA sample, polymerase, DNA nucleotides, primer

Biotechnology (5 decks)

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