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DNA manipulation Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

restriction endonuclease

A

endonucleases that target a specific recognition site (restriction site)

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2
Q

DNA ligase

A

enzymes that anneals 2 fragments of DNA.
- form phosphodiester bonds

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3
Q

DNA polymerase

A

add nucleotides to DNA
- require a primer

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4
Q

taq polymerase

A

a heat resistant polymerase required to bind new nucleotides to single stranded DNA
-ideal temp: 72 degrees celcius

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5
Q

blunt ends

A

(restriction enzymes create) a straight cut through DNA with no overhanging nucleotides

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6
Q

sticky ends

A

(restriction enzymes create) a staggered cut through DNA which results in overhanging nucleotides

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7
Q

CRISPR in bacteria

A
  • primative adaptive immune system
  • defence mechanism
  • used to protect against bactertiophage infection by cutting viral DNA preventing them from replicating
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8
Q

PAM (protospacer adjacent motif)

A

short sequence of nucleotides that Cas1 and Cas2 recognise.
they are signalled to extract the protospacer by cutting viral DNA just before the PAM.

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9
Q

Cas1 & Cas2

A

run along viral DNA looking for a PAM sequence. cut out a short section of DNA called the protospacer, just before the PAM sequence.
(CRISPR associated enzymes)

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10
Q

protospacer

A

section of viral DNA cut out by Cas1 and Cas2 before being inserted into the CRISPR gene

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11
Q

spacer

A

short sequences of viral DNA that are inserted into the CRISPR sequence

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12
Q

repeat

A

palindromic repeats of DNA sequences in between spacers in the CRISPR array

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13
Q

CRISPR array

A

contains repeats and spacers. part of the CRISPR array is (spacer and part of repeat) is turned into gRNA

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14
Q

gRNA (guide RNA)

A

CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it.
specific sequence determined by CRISPR to guide Cas9 to a specific site.

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15
Q

sgRNA (single guide RNA)

A

guide RNA created by scientists.
to instruct Cas9 to cut a specific site (when using CRISPR-Cas9 in gene editing)

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16
Q

Cas9

A

endonuclease that creates a blunt cut at a site specified by gRNA

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17
Q

CRISPR-Cas9

A

a complex formed between gRNA and Cas9 which can cut a target sequence of DNA.
bacteria defence mechanism against viruses
used fro genome editing by scientists

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18
Q

why is DNA altered after Cas9

A

when the DNA is cut, enzymes will try to repair it.
repair mechanisms are prone to errors so Cas9 will continue to cut that target DNA until a mutation occurs. the gene is now non-functional.

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19
Q

gene knock in

A

to add DNA segments.
scientists add them to the cell in hope they will be used in the post-cut repair process.

20
Q

gene knock out

A

scientists prevent the expression of a target gene to understand its funtion in an organism

21
Q

PCR (polymerase chain reaction)

A

used to amplify DNA by making identical copies

22
Q

purpose of PCR

A

used whenever there I’d an insufficient DNA sample for testing
- analysing gene fragments for genetic testing
- paternity testing
- forensic testing

23
Q

materials required for PCR

A
  • DNA sample
  • taq polymerase
  • source of nucleotides
  • DNA primers
    (all placed in a thermocycler)
24
Q

PCR stage 1

A

Denaturation:
- DNA sample is heated to 90-95 degrees celcius
-hydrogen bonds break to form single stranded DNA

25
Q

PCR stage 2

A

annealing:
- single stranded DNA is cooled to 50-55 degrees celcius
- allows primers to bind

26
Q

PCR stage 3

A

elongation:
- DNA is heated to exactly 72 degrees celcius, optimal temp. of taq polymerase
- taq polymerase binds to the primers and synthesises new strand of DNA
(cycle is repeated 35-45 times)

27
Q

purpose of primers in PCR

A

forward primer: binds to the start codon at the 3’ end of the template strand
reverse primer: binds to the stop codon on the 3’ end of the coding strand
Allows taq polymerase to synthesise new DNA strand.

28
Q

applications of gel electrophoresis

A

used to measure the size of DNA fragments.
- can be used to detect genetic disorders like cystic fibrosis
- can be used for DNA profiling by comparing STRs

29
Q

factors affecting how a DNA fragment will run

A
  • voltage
  • gel composition
  • buffer concentration
  • time
30
Q

STRs (short tandem repeats)

A

small sections of repeated nucleotides that vary in length between people

31
Q

gel electrophoresis steps

A
  1. DNA samples are placed in wells at one end of the gel
  2. an electric current is passed through the gel
  3. smaller fragments of DNA move faster/travel further through the gel
  4. DNA is stained to make bands visible
32
Q

why is a standard ladder used when reading gels

A

standard ladder contains DNA of a known length.
it is compared to the DNA samples being analysed to estimate their length

33
Q

plasmid

A

small circular loop of DNA separate from the chromosome.
typically found in bacteria.

34
Q

recombinant plasmid

A

circular DNA that is ligated to incorporate a gene of interest

35
Q

bacterial tranformation

A

the process by which bacteria take up foreign DNA from their environment.
used to introduce recombinant plasmids into bacteria

36
Q

reporter gene

A

gene with an easily indentifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest

37
Q

electroporation

A

delivering an electric shock to bacterial membranes to increase membrane permeability, and increase the likelihood of bacterial transformation

38
Q

heat shock

A

bacteria and plasmids are placed in a calcium ion solution on ice. it is then rapidly heated and cooled to increase membrane permeability, and increase the likelihood of bacteria transformation

39
Q

lacZ enzyme

A
  • lacZ gets inserted into the recombinant plasmid, already containing the insulin subunit gene.
  • lacZ produces beta-galactosidase
40
Q

beta-galactosidase

A
  • creates a fusion protein with the insulin subunit protein which protects it from being digested by the cell.
  • identifiable marker because it turns x-gal blue when plated with it. signals it has taken up the recombinant plasmid
41
Q

how insulin is isolated from bacteria

A
  • cyanogen bromide solution is added to bacteria
  • breaks down methionine at the start of the insulin gene, separating it from the beta galactosidase
  • can now be isolated and purified
42
Q

genetic modification

A

the manipulation of an organisms genetic material using biotechnology

43
Q

cisgenesis

A

process of inserting DNA from the same species

44
Q

transgenesis

A

process of inserting DNA from another species

45
Q

how a transgenic plant is made

A
  1. desired trait is identified and isolated
  2. gene of interest is inserted into the plant cell
  3. transformed plant cell is grown repeatedly in a lab before being transferred to fields

biology (3 decks)

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