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Grade 12 Biology > Biotechnology Part 1 > Flashcards

Biotechnology Part 1 Flashcards

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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

23
Q

what is a sticky end

A

a signle-stranded end of the restriction fragment

24
Q

what is a blunt end

A

straight ends without any single-stranded regions

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

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

27
Q

plasmid

A

extisting circular bacterial DNA

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
29
Q

genome

A

an organisms’ complete set of DNA

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

A

repeats and spacers

35
Q

what does CRISPR

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 is the Cas gene

A

CRISPR associated genes that are responsible for making the CRISPR locus and carryong out most of the slides

38
Q

what are the two main steps in CRISPR/Cas9 mechanism

A

A. Immunization
B. Adaptive/Acquired Immune Response

39
Q

what are the steps in the CRISPR/Cas9 mechanism 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

40
Q

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

A

acquiring foreign DNA

41
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
42
Q

what is a repeat?

A

palindromic sequence same sequence repeated between each spacer

43
Q

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

A

transcribe to RNA

44
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

45
Q

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

A

Processing (cutting) RNA

46
Q

Describe the processing of RNA

A

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

47
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

48
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

49
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

50
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

51
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
52
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

53
Q
A

Grade 12 Biology (26 decks)

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