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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
Q

Uses of DNA Barcoding

A

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
Q

What Can DNA Barcoding Not Answer

A

Identifying hybrid species, single gene regions, or closely related species

27
Q

DNA Regions Amplified in Plants, Animals, and Fungi

A

Plants rbcL, chloroplast, MatK gene
Animals CO1, mitochondrial gene
Fungi ITS, nuclear intron near ribosomal RNA gene
Conserved but variable

28
Q

Highly Conserved But Variable

A

A segment of DNA that remains similar across different species but also polymorphic

29
Q

Purpose of Barrier Filter Tips on the Micropipettes

A

Avoids cross-contamination, keeps aerosol out

30
Q

Why Use a Very Small Piece of DNA To Perfrom Extraction On

A

Large samples introduce cellular components and debris that can inhibit PCR

31
Q

Different Buffers Usage

A

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
Q

What Was Added to PCR Tube for PCR

A

Master mix contains DNA nucleotides and polymerase
Corresponding primer for specific type of organism
Primer and DNA were added seperately

33
Q

Electropherogram

A

Visual representation of your sequence data where each nucleotide is assigned a different color by the computer software

34
Q

QS Score

A

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
Q

Phred Score

A

Quality score for each individual nucleotide
Accuracy of the nucleotide call
20 is the cut-off for a high-quality sequence

36
Q

Peak Quality

A

Clear, high peaks with little to no background noise mean high quality Background noise refers to many overlapping peaks in same location

37
Q

BLAST

A

Quickly identify any close matches to your sequence in sequence databases

38
Q

MUSCLE

A

Align your sequences of DNA Barcodes to visually represent data between two or more sequences

39
Q

Lines and Grey Areas in a DNA Barcode

A

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
Q

Sanger Sequencing/Chain Termination

A

Fredrick Sanger
Sequences small sections of DNA
Replicates fragments of DNA using PCR

41
Q

Shotgun Sequencing

A

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
Q

Sequencing by Synthesis/SBS

A

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
Q

Nanopore Sequencing

A

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
Q

Enzymes Involved in DNA Replication

A

Helicase, binding proteins, primase, DNA polymerase III and I, and ligase

45
Q

Helicase

A

Unzips DNA

46
Q

DNA Polymerase

A

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
Q

Steps of DNA Replication

A
  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
Q

Reading DNA

A

Basis of orientation of sugar phosphate backbone
5′ end has free phosphate group whereas 3′ end has free hydroxyl group

49
Q

When Does DNA Replication Happen Naturally

A

Prior to cell division during the synthesis phase of the cell cycle

50
Q

After 20-30 Cycles How Many Copies of the Original DNA Sequence Has Been Made

A

1 million

51
Q

Primer Dimer

A

A small band that runs ahead of the other bands
Formed when primers bond to each other and replicate themselves

52
Q

Conversions

A

1uL = a millionth L
1 mL = 1000 uL
1 L = 1000 mL
1L = 1,000,000 uL

53
Q

Why is DNA Temperature Sensitive

A

Buffers have different jobs adn DNA needs to be released without being denatured

54
Q

DNA Throughout the Extraction Process

A

Wash buffer bound to beads washed out
DI water new tube released dna from beads
Spin column sometimes above beads sometimes in beeds

55
Q

Role of Supernatant and Matrix

A

Supernatant DNA
Matrix DNA was bound to matrix so DNA wouldn’t get washed through

56
Q

Sequencing Used and Why

A

Sagar sequencing to sequence one gene
Shotgun is using sangar but every gene
Nanopore too expenxive
Synthesis special equipment

57
Q

CRL Score

A

How long the computer could read the sequence in base pairs

58
Q

Role of Primers and Primase in DNA Replication

A

Tells polymerase where to go primase places primer

59
Q

Primer Dimer But No Other Bands Meaning

A

PCR worked but they didn’t extract DNA

60
Q

What’s in Sample of DNA to Replicate Via PCR

A

DNA sample, polymerase, DNA nucleotides, primer

Biotechnology (2 decks)

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