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Grade 12 Biology > Biotechnology Part 1 > Flashcards

Biotechnology Part 1 Flashcards

1
Q

what does DNA amplification do? Why?

A

it create millions of copies of a specific DNA sequence to increase the amount of DNA

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2
Q

what are the materials in PCR?

A
  • DNA template
  • nucleotides
  • DNAP (Taq polymerase)
  • DNA primers
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3
Q

what are the 3 steps of the PCR cycle?

A
  • Denaturation
  • Annealing
  • Extension
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4
Q

what is denaturation in the PCR cycle

A

temperature is increased to separate DNA strands

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5
Q

what is the annealing in the PCR cycle

A

temperature is decreased to allow primers to base pair to complementary DNA template

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6
Q

what is the extension in the PCR cycle

A

plymerase extends primer to form nascent DNA strand

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7
Q

describe a primer in the PCR cycle?

A
  • a synthetic sequence (artificial)
  • single stranded DNA
  • provides the 3’ OH for polymerization
  • 2 primers used for complementary to each end of the target region of the DNA
  • defines length of target DNA to be replicated
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8
Q

Why is Taq used in the PCR cycle?

A

the temp of the PCR denatures the enzymes meaning they don’t work so something else must be used

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9
Q

what was done before Taq was used?

A
  • 3 water baths at 3 temperatures with a re-addition of DNAP at the beginning of each synthesis step
  • tedious and time consumming as it had to be done manually all day
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10
Q

describe the thermal cycler

A

has a plate that heats and cools in a process that takes 1-2 hours

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11
Q

what is used for unwinding compare natural DNA replication and PCR replication.

A

natural: Ori on DNA template + helicase

PCR: template + heat

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12
Q

what is used for priming? compare natural DNA replication and PCR replication.

A

natural: RNA primer + primase

PCR: DNA primers x2 + annealing

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13
Q

what is used for elongation? compare natural DNA replication and PCR replication.

A

natural: nucleotides + DNAP

PCR: nucleotides + Taq polymerase

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14
Q

what is used for termination? compare natural DNA replication and PCR replication.

A

natural: end of chromosome OR meets another replication bubble

PCR: end of DNA template or a change in temp

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15
Q

at which cycle do you get the correctly sized target sequence?

A

2 round of replication

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16
Q

what does endonuclease do?

A

breaks phosphodiester bonds within a nucleotide chain

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17
Q

what is a restriction enzyme?

A

a biological ‘scissor’

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18
Q

what is the function of a restriction enzyme

A

acts like the immune system of bacteria to protect it against DNA from other organisms

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19
Q

what does a restriction enzyme do in bacteria?

A

recognizes short nucleotide sequences in the foreign DNA - cuts cvalent phosphodiester bonds of both strands of DNA rendering foreign DNA harmless

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20
Q

restriction site

A

sequene recognized and cut by restriction enzyme

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21
Q

what does palindromic mean in bio?

A

same sequence on complementary strand in opposite orientation

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22
Q

what is a restriction fragment?

A

pieces of DNA created by restriction enzymes

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23
Q

what is a sticky end

A

a signle-stranded end of the restriction fragment

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24
Q

what is a blunt end

A

straight ends without any single-stranded regions

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25
Q

how does naming restriction enzymes work?

A

1 - first letter of first word in organism name
2 - first two letters of second word in organism name
3 - stran
4 - roman numeral of enzyme in this strain

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26
Q

recombinant DNA

A

genes from two different sources (often different species) combined into one molecule - any DNA cut with the same restriction can be ligated together because they have the same sticky ends that are complementary

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27
Q

plasmid

A

extisting circular bacterial DNA

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28
Q

what are the general steps of forming a recombinatant

A
  • restriction enzyme digestion of plasmid and gene of interest
  • hybridization of matching sticky ends on gene of interest and plasmid
  • DNA ligase seals gene of interest with plasmid
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29
Q

genome

A

an organisms’ complete set of DNA

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30
Q

human genome project

A

is an international collavorative research effort 1998-2003 to sequence and map all genes in human beings

31
Q

why does the CRISPR (Cas 9 system exist)

A

naturally as the bacteria’s immune system to destroy phages by recognizing foreign DNA and making a double stranded cut at that DNA

32
Q

how does the CRISPR mechanism work

A
  • CRISPR locus is transcribed and translated to make the CRISPR/CAs9
  • CRISPR RNA targets Cas9 to specific complementary region on foreign DNA
  • Cas9 makes a double stranded break at that location
33
Q

what is the CRISPR/Cas9 structure?

A

ribonucleoprotein complex with CRISPR RNA and Cas9 endonuclease

34
Q

what are the two main parts of the CRISPR locus

A

repeats and spacers

35
Q

what does CRISPR stand for?

A

clustered regularly interspaced short palindromic repeats

36
Q

what are the characteristic of a CRISPR locus

A
  • regularly spaced
  • short sequence
  • identical
  • palindromic
37
Q

what are the two main steps in CRISPR/Cas9 mechanism

A

A. Immunization
B. Adaptive/Acquired Immune Response

38
Q

what are the steps in the CRISPR/Cas9 mechanism

A

A. Immunization
1. acquire foreign DNA
2. Transcribe into RNA
3. process (Cut) RNA
4. incorporate into Cas9 protein
B. adaptive/acquired immune response
1. Targeting by cRNA
2. Interferene/Inactivation

39
Q

What is the 1st step of the CRISPR/Cas9 mechanism

A

acquiring foreign DNA

40
Q

describe acquiring foreign DNA

A
  • bacteria would get infected by viruses
  • sometimes bacteria survive infection and takes the viral DNA placing it within the CRISPR locus (spacer)
  • is followed by a new repeated sequence
41
Q

what is a repeat?

A

palindromic sequence same sequence repeated between each spacer

42
Q

What is the 2nd step of the CRISPR/Cas9 mechanism

A

transcribe to RNA

43
Q

What is transcription to RNA in CRISPR

A

the CRISPR locus transcribed into RNA called pre-cRNA, which causes palindromic nature of the repeat forms hairpin loops

44
Q

what is the third step of the CRISPR/Cas9 mechanism?

A

Processing (cutting) RNA

45
Q

Describe the processing of RNA

A

pre-cRNA cut into pieces forming crRNA with each one containing one spacer

46
Q

what is the fourth step of the CRISPR/Cas9 mechanism? Describe it

A

incorporating into Cas9 protein - the crRNA is placed into Cas9 protein

47
Q

How does Adaptive/Acwuired Immunity work?

A

if the virus tries to infect the cell again, the crRNA/Cas9 already exists SOOOO:
- the crRNA binds to the complmentary viral DNA
- causes a confirmational change in Cas9 activating it’s endonuclease acitivity
- Cas9 makes a double stranded blunt end that breaks and destroys the viral DNA

48
Q

What is the ‘power’ of CRISPR /Cas9

A

you can manipulate the crRNA sequence to target whatever you want to be cut by Cas9

49
Q

what is gRNA? what does it do?

A

guide RNA - replaces the crRNA with an RNA sequence of choice that is complementary to a desired DNA region or gene of interest - it will direct the Cas9 complex to the target site

50
Q

what is PAM? what does it do?

A

protospare Adjacent Motif a sequence where N is any base

  • Cas9 recognizes and binds to any PAm and unwindes that region of DNA
51
Q

how does the Cas9 Endonuclease work?

A

if the exposed signle stranded DNA region unqound by Cas9 is complementary to the gRNA they will base pair with causes a conformational change in Cas9 activating its endonuclease activity, which results in a double stranded break at a site upstream of PAM

52
Q

how does CRISPR/gRNA apply to gene editing?

A

gRNA sequence can be used, then any DNA region can be targeted and cut by Cas9, making gene editing more precise and less time-consuming

53
Q

what are the two main repair mechanisms?

A

HDR: homology directed repair
NHEJ: non-homologous end joining

54
Q

what is gene editing - repair by insertion

A

placement of a new DNA sequence at the targeted region

55
Q

describe homology directed repair

A

cell with cut DNA is subjected to a large dose of the donor template DNA and natural DNA-repair mechanisms of the cell will insert the desired genetic material

56
Q

what does the donor DNA sequence consist of?

A
  • desired sequence
  • DNA homologous to the blunt ends of the cut DNA flanking the desired sequence
57
Q

what is the power of Power of CRISPR & HDR

A

you can insert any DNA sequence you want at any specific location

58
Q

describe NHEJ

A

double-stranded breaks are ligated back together without the need for a homologous DNA template, which can result in missing bases

59
Q

what is the Power of CRISPR and NHEJ

A

due to the error prone nature of NHEJ, you can target any gene and inactivate it

60
Q

what is gel electrophoresis?

A

size separation of nucleic acids by moving them through a gel medium using electric current

61
Q

how is the gel in gel electrophoresis made?

A
  • liquid solution of gel medium is poured into a mold and allowed to set and solidify
  • a cone is placed at one end of the gel, when removed it creates the wells where DNA is loaded
62
Q

describe the liquid buffer in the gel

A
  • contains ions to allow for flow of electricity
  • prevents the gel from overheating and dryingout
63
Q

what are the two gel mediums?

A

agarose and polyacrylamide

64
Q

describe agarose

A
  • seaweed extract
  • lower resolution resolving power and
  • separation of nucleic acid
65
Q

describe polyacrylamide

A
  • artificial polymer
  • higher resolution resolving power
  • nucleic acid and protein separation abilities
66
Q

resolving power

A

the more precisely closely sized DNA fragments can be distinguished

67
Q

describe the movement of DNA within the gel

A

DNA is negatively charged due to the phosphates so it will move toward the positive electrode

SHORT:
- moves through gel easily and travels further
LONG:
- moves through gel slower and not as far as the size makes it get caught int he web of polymers

68
Q

what are the components of the loading dye

A

glycerol and coloured dyes

69
Q

what is the purpose of glycerol in the loading dye?

A
  • adds density to DNa sample
  • so makes it sink down to the bottom of the well when placed int he gel
  • DNA isn’t as easily lost in the buffer or spilled
70
Q

what is the purpose of coloured dyes?

A
  • used for tracking distance travelled on the gel by DNA since DNa is invisble
  • indicates when it is time to turn off the gel electrophoresis
71
Q

what is ethidium bromide? Describe it.

A
  • a chemical that binds to the DNA
  • makes DNA visible
  • not good for the body - carcinogenic
  • glows under UV light
72
Q

Describe SYBR Safe

A

not carcinogenic and glows green

73
Q
A

Grade 12 Biology (30 decks)

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