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Biology 3/4 > chap 3- DNA manipulation techniques and applications > Flashcards

chap 3- DNA manipulation techniques and applications Flashcards

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1
Q

what are restriction endonucleases

A

enzymes that cut DNA at a specific recognition site

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2
Q

what are restriction enzymes used for

A

to cut DNA into smaller more usable fragments and isolate particular regions of interest (single genes)

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3
Q

When do restriction enzymes occur naturally

A

in bacteria

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4
Q

what do restriction enzymes form

A

bacterial cells defense system, targeting foreign DNA that may enter the cell

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5
Q

What are sticky-ends

A

when restriction enzymes leave DNA fragments with overhanging ends

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6
Q

what are blunt ends

A

when restriction enzymes leave clean-cut ends

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7
Q

how do sticky end restriction enzymes cut the DNA backbone

A

at a different location on each strand within the recognition site

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8
Q

how do blunt end restriction enzymes cut the DNA backbone

A

by cutting the sugar-phosphate backbone on both strands of the DNA molecule at the same location within the recognition site

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9
Q

What are ligases

A

a group of enzymes that join fragments of DNA or RNA and create a phosphodiester bond between them.

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10
Q

What are polymerases

A

enzymes that catalyse the formation of polymers, in particular nucleic acids

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11
Q

why does DNA polymerase act

A

to produce identical copies of DNA

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12
Q

what does RNA polymerase do

A

acts to assemble RNA. In cells, RNA polymerase synthesizes mRNA, rRNA and tRNA by transcription of genes.

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13
Q

what does PCR stand for

A

Polymerase Chain Reaction

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14
Q

what is PCR used for

A

to obtain larger amounts of DNA when there is only a small amount

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15
Q

In PCR: what does the DNA sample provide

A

a target sequence of DNA that is to be occupied and amplified.

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16
Q

In PCR what do primers provide

A

a starting point for DNA to be built on

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17
Q

what are primers

A

short nucleic acid sequences

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18
Q

In PCR what are free nucleotides used for

A

added to solution in order for DNA to be built

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19
Q

In PCR what does taq polymerase do

A

binds to primer and begins building DNA from the free nucleotides

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20
Q

In PCR what is the buffer solution for

A

provides suitable chemical environment for activity of polymerase

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21
Q

what is the significance of Taq polymerase for PCR

A

its extracted from bacteria that lives in hot springs meaning it can function in high temps without degrading

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22
Q

What are the 3 steps of PCR

A

denaturation. annealing. extension

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23
Q

what happens during denaturation

A

sample heated to 95 degrees. breaks hydrogen bonds between the strands of DNA to obtain single strands of DNA.

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24
Q

what happens during annealing

A

temp is reduced to 50-60 degrees. allows primers to bind to complementary base sequences through hydrogen bonds

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25
Q

what happens during extension

A

temp increased to 72 degrees. allows taq polymerase to attach to the primers on the DNA strands. the taq polymerase moves along each strand adding free nucleotides to form double stranded DNA.

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26
Q

in each PCR cycle how much is the number of double stranded copies of the DNA increased by

A

double the amount

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27
Q

what does gel electrophoresis do

A

separates fragments of negatively charged DNA by length

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28
Q

what can gel electrophoresis technique be used for

A

DNA screening, fingerprinting for forensics, paternity cases, confirming correct gene has been amplified by PCR

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29
Q

what does DNA profiling do

A

identifies one individual from another individual

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30
Q

what is DNA profiling used for

A

identifying perpetrators of a crime, identifier of bodies and to settle paternity disputes

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31
Q

what are non-coding DNA sections

A

regions that are not transcribed or translated into a protein

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32
Q

what does STR stand for

A

short tandem repeats

33
Q

what does DNA profiling rely on

A

an individuals unique DNA, specifically the non-coding sections of DNA which vary widely between individuals

34
Q

what are STRS

A

sections of 2-6 bases located within the non-coding sections of DNA, located on specific chromosomes can be used to identify a person uniquely from someone else

35
Q

what is the process of DNA profiling

A

DNA sample extracted using restriction enzymes
STR’s amplified using PCR with specific primers for each STR
Gel electrophoresis differentiates STR’s
DNA sample is a match if lengths of particular STR’s are all the same size

36
Q

what does CRISPR stand for

A

Clustered regularly interspaced short palindromic repeats

37
Q

What does regularly interspaced mean

A

spaces occur at regular intervals

38
Q

what does palindromic mean

A

short sequence of letters that reads the same forwards and backwards

39
Q

What is spacer DNA

A

sections of DNA from foreign substances such as viruses, bacteriophages or plasmids that have been incorporated into the bacterium’s genome.

40
Q

what are Cas genes

A

genes that encode Cas proteins which are helicases that cause DNA to unwind enabling endonucleases to cut the DNA

41
Q

what is adaptive immunity

A

immunity an organism acquires after exposure to a pathogen

42
Q

what is a bacteriophage

A

a type of virus that infects its host bacterial cell by injecting its genetic material into the host cell. takes over host cell until it kills the cell

43
Q

what is the role of Cas1 proteins

A

break apart bacteriophage DNA and store a section of that DNA in the CRISPR locus

44
Q

What is the bacterial CRISPR immune system

A

DNa is transcribed to make tracrRNA and pre-crRNA along with ranscribing and translating Cas proteins

45
Q

what does CRISPR array consist of

A

duplicate sequences of palindromic repeats belonging to the bacterial genome flanked by spacers belonging to foreign genetic material

46
Q

what is pre-CRISPR RNA (pre-crRNA)

A

RNA with repeat palindromes and spacers that is transcribed in one strand

47
Q

what is tracer RNA (tracrRNA)

A

has sections that are complimentary to the palindrome repeats enabling it to anneal to the pre-crRNA strand within the Cas9 protein

48
Q

how does Cas9 complex work to defend bacteria from viral DNA

A

nuclease enzyme will search for a short specific sequence unique to viral genome (PAM). then the viral DNA will unwind. one target DNA is located endonuclease will snip both strands of DNA just a few bases upstream from the PAM

49
Q

what does PAM stand for

A

protospacer adjacent motifs

50
Q

where is PAM found

A

after the DNA sequence that the Cas9 nuclease wants to cut

51
Q

What do PAMs do

A

help bacteria distinguish their own DNA from viral DNA

52
Q

what is guide RNA (sgRNA)

A

synthetic version of the DNA strand being fed through Cas9

53
Q

what are some CRISPR applications

A

effective with helping sickle cell anaemia, cystic fibrosis, muscular dystrophy

54
Q

What are plasmids

A

small circular pieces of double stranded extrachromosomal DNA molecules that are physically separated from chromosomal DNA and can replicate independently of chromosomal DNA.

55
Q

where are plasmids found

A

bacteria

56
Q

what is a recombinant plasmid

A

a plasmid that has been altered in the laboratory through the insertion of a foreign gene

57
Q

what is the origin of replication

A

a DNA sequence that allows initiation of replication within a plasmid through the use of host cells replication machinery

58
Q

What is an antibiotic resistance gene

A

allows for selection of plasmid containing bacteria

59
Q

what is a selectable marker

A

enables selection of transformed plasmids by differentiating them from non-transformed plasmids

60
Q

what is a promotor

A

DNA sequence upstream from gene to be expressed that RNA polymerase attaches to and begins protein synthesis of that gene

61
Q

what are restriction sites

A

specific DNA sequences that act as cut sites for restriction enzymes

62
Q

what is the inserted gene

A

gene that has been inserted into plasmid

63
Q

what is transformation

A

process used by bacterial cells to introduce foreign material into the cell, this foreign material can form into a plasmid

64
Q

what is a vector

A

any vehicle that is used to ferry a desired DNA sequence into a host cell as part of a molecular cloning procedure

65
Q

what is complementary DNA (cDNA)

A

a DNA copy of a mature mRNA molecule produced by reverse transcriptase using a reverse transcriptase enzyme

66
Q

how is cDNA made

A

from mature mRNA cause its had its introns removed and therefore only contains exons

67
Q

What are the two ways you can transform bacterial cells

A

heat shock and electroporation

68
Q

what is electroporation

A

where a pulse of electricity is used to briefly open pores in the cell membrane of bacteria so plasmids can be taken up into the bacteria

69
Q

what is heat shock treatment

A

bacterial cells and plasmids placed in ice-cold solution containing calcium ions. temp is rapidly increased creating pressure difference between out/inside of cell inducing the formation of pores in the plasma membrane and allowing the supercoiled plasmid to enter. bacteria is then allowed recovery time

70
Q

how can recombinant plasmids be identified

A

bacteria cultures are placed onto agar plates with an antibiotic that recombinant plasmids are resistant to. bacterial cells that have been transformed through the uptake of the recombinant plasmid will survive, the ones that haven’t will die

71
Q

What is a GMO

A

genetically modified organism

72
Q

what is a transgenic organism

A

an organism that’s genome has been altered through the insertion of a gene from another type of organism

73
Q

what is insulin

A

a peptide hormone that promotes the uptake of sugar from the bloodstream and its storage in muscle or adipose tissue.

74
Q

what produces insulin

A

B-cells of islets of Langerhans of the pancreas

75
Q

what does insulin consist of

A

two polypeptide chains, an A chain consisting of 21 amino acids and a B chain consisting of 30 amino acids

76
Q

what happens during option 1: placement of insulin gene within beta-galactosidase (B-gal)

A

when b-gal is functioning, bacterial colonies that are plated on a medium of x-gal will be blue in colour. when insulin is placed within the B-gal gene, the B-gal isn’t produced and therefore cant break down X-gal and bacterial colonies that have taken up the recombinant plasmid containing the insulin gene will be white in colour allowing for easy identification

77
Q

what happens in option 2: placement of insulin gene next to B-gal

A

promotes transcription of the insulin gene, which then allows for the production of functional insulin.
this is because when lactose is added to the E.Coli cell it promotes the synthesis of B-gal and cause the insulin gene is next to the B-gal gene, insulin gets synthesized as well

78
Q
A

Biology 3/4 (22 decks)

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