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Biochemistry BCC > Recombinant DNA Technoloy 1 > Flashcards

Recombinant DNA Technoloy 1 Flashcards

1
Q

What is Histone?

A

A protein that provides structural support for a chromosome.

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2
Q

A protein that provides structural support for a chromosome.

A

Histone

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3
Q

Name the 20 amino acids.

A

Essential:
histidine,
isoleucine,
leucine,
lysine,
methionine,
phenylalanine,
threonine,
tryptophan,
valine.

Nonessential:
alanine,
asparagine,
aspartic acid
glutamic acid
serine.

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4
Q

What is genome sequencing?

A

a comprehensive list of every base pair sequence of DNA.

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5
Q

what is homogenisation?

A

A process that involves breaking apart cells - releasing organelles and cytoplasm.

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6
Q

A process that involves breaking apart cells - releasing organelles and cytoplasm.

A

Homogenisation

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7
Q

From the Greek root…… means “of the same kind.

A

homogenes

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8
Q

How is DNA prepared for (?)

A
  1. Get my sample from the animal
  2. homogenise the cells or tissues
  3. use classic phenol extraction/centrifugation
  4. the sample will separate into an aqueous layer and phenol layer.
  5. the aqueous layer will contain RNA nad DNA ad the phenol layer will contain protein.
    6 - Ethanol precipitation - purifying or concentrating DNA, RNA, Polysaccharides.
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9
Q

What is DNA recombination?

A

The process during which a stretch of DNA recombines and produces a new allelic combination

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10
Q

what is phenol extraction?

A

Separating nucleic acids from other cellular substances

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11
Q

what is the process of Separating nucleic acids from other cellular substances called?

A

Phenol Extraction

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12
Q

Define: phenol

A

any of a family of organic compounds characterized by a hydroxyl (―OH) group attached to a carbon atom that is part of an aromatic ring.

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13
Q

How many layers are there in the sample after a phenol extraction/cetrifugation? and what are they?

A

Aqueous Layer

phenol layer

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14
Q

What substance is contained in an aqueous layer after phenol extraction/centrifugation?

A

DNA + RNA

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15
Q

What substance is contained in a phenol layer after phenol extraction/centrifugation?

A

Proteins

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16
Q

What is ethanol precipitation?

A

A method used to purify and/or concentrate RNA, DNA, and polysaccharides such as pectin and xyloglucan from aqueous solutions by adding ethanol as an antisolvent.

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17
Q

Why is ethanol used on DNA extraction?

A

Ethanol precipitation is often used to obtain DNA molecules extruded from cells.

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18
Q

_________ __________ is often used to obtain DNA molecules extruded (thrust, forced out) from cells.

A

Ethanol precipitation

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19
Q

Name 3 uses of Type II Restriction Endonucleases

A
  1. Gene cloning
  2. DNA fragmentation
  3. DNA analysis
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20
Q

What is DNA fragmentation?

A

The separation or breaking of DNA strands into pieces

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21
Q

Gene cloning, DNA fragmentation and DNA analysis are three ways in which ___ __ ___________ _____________ can be usedin DNA extraction.

A

Type II Restriction Endonucleases

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22
Q

Define: nuclease

A

An enzyme that cleaves the chains of nucleotides in nucleic acids into smaller units.

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23
Q

How many types of Type II Restriction Endonucleases are recognised?

A

over 3500

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24
Q

How many different DNA sequences do the 3500+ Type II enzymes recognise?

A

350+

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25
Q

Why are type I restriction enzymes preferable to type II?

A

Type I will randomly cut DNA, at times far from the recognition site. Type II will cut DNA from the recognition site at the defined position..

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26
Q

Which answer is correct about type I and type II restriction enzymes:
A) Type II will randomly cut DNA, at times far from the recognition site. Type I will cut DNA from the recognition site at the defined position.
B) Type I will randomly cut DNA, at times far from the recognition site. Type II will cut DNA from the recognition site at the defined position.
C) Type I will randomly cut the recognition site, close to the DNA. Type II will cut DNA from the recognition site at the defined position.

A

B) Type I restriction enzymes will randomly cut DNA, at times far from the recognition site. Type II restriction enzymes will cut DNA from the recognition site at the defined position..

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27
Q

True or false: Type I restriction enzymes will randomly cut DNA, at times far from the recognition site. Type II restriction enzymes will cut DNA from the recognition site at the defined position..

A

True

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28
Q

True or false:
Type II restriction enzymes will randomly cut DNA, at times far from the recognition site.

A

False

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29
Q

True or false:
Type II will cut DNA from the recognition site at the defined position..

A

True

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30
Q

What is EcoRI?

A

A restriction endonuclease enzyme isolated from species E. coli

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31
Q

A restriction endonuclease enzyme isolated from species E. coli

A

What is EcoRI.

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32
Q

What does EcoR1 do in genetic engineering?

A

Scans the DNA sequence for a specific base letter sequence code: GAATTC. When it finds this code, the enzyme cuts the plasmid at this point, leaving a sticky end behind
which helps new genes to attach. Dna ligase comes along and stiches the ends otgether.

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33
Q

What is a palindrome?

A

a word, phrase, or sequence that reads the same backwards as forwards. In DNA; anti parellel sequence:

5’ G C G C 3’
3’ C G C G 5’

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34
Q

A word, phrase, or sequence that reads the same backwards as forwards (An anti parallel DNA sequence)

A

Palindrome

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35
Q

What are the two manners of cut to target DNA that are useful in genetic engineering?

A

Blunt Cut - They cut straight across the DNA, leaving no exposed unpaired bases

Sticky end cut - the type II enzyme cuts the DNA at a specific site (GAATTA) and leaves a sticky end behind which the new piece of DNA is stitched together by the DNA ligase.

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36
Q

What is the difference between a sticky end cut and a blunt end cut?

A

Sticky ends cuts by type 2 restriction endonucleases leave a stick end in which DNA ligase will bind the new DNA chain to the cut one.

Blunt end cuts leave no exposed unpaired bases.

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37
Q

A ______ ___ cuts by type 2 restriction endonucleases leave a stick end in which DNA ligase will bind the new DNA chain to the cut one.

A

Sticky End

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38
Q

A _____ ___ cut leave no exposed unpaired bases.

A

Blunt end

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39
Q

Why is it called a sticky end cut?

A

Because a single strand of DNA is left behind, which can form a base pair with the sticky end of a complimentary DNA molecule.

40
Q

This orthodox Type IIP enzyme, binds to DNA in the absence of metal ions and cleaves the DNA sequence 5′-GGTAC^C-3

A

Kpn1

41
Q

True of false: A Kpn1 enzyme cut produces a blunt end cut with no overhangs

A

False.

42
Q

True of false: A Kpn1 enzyme cut produces sticky end cut with overhang

A

True

43
Q

True of false: An EcoRV enzyme cut produces sticky end cut with overhang

A

False

44
Q

True of false: A, EcoRV enzyme cut produces a blunt end cut with no overhangs

A

True

45
Q

How often will recombination endonucleases cut?

A

every 44 (256) bases, while a 6 bp cutter cleaves every 46 (4096) bases.

46
Q

True of false:
Restriction enzymes with shorter recognition sequences cut more frequently than those with longer recognition sequences

A

True

47
Q

In the digestion frequency ofrecombination endonucleases, what does 4n represent?

A

The number of bases G/A/C/T

48
Q

In the digestion frequency of recombination endonucleases, what does ‘n’ represent?

A

The length of recognition sequence.

49
Q

How many enzyme cuts are made in IN n=4?

A

256 bp (base pairs)

50
Q

How many enzyme cuts are made in IN n=6?

A

4096 bp (base pairs)

51
Q

What is DNA ligation?

A

Joining of DNA strands together by catalysing the formation of a phosphodiester bond

52
Q

Name the 3 steps of DNA ligation

A
  1. Self adenylation - a process in which an adenosine monophosphate molecule is covalently attached to the amino-acid side chain of a protein
  2. Adenyl group transfer to DNA
  3. Phosphodiester bond forms
53
Q

Molecular basis of DNA ligation

A

DNA ligases catalyse the formation of phosphodiester bonds at single-strand breaks between adjacent 3′-hydroxyl and 5′-phosphate termini in double-stranded DNA

54
Q

___ _______ catalyse the formation of _____________ bonds at ______-strand breaks between adjacent 3′-hydroxyl and 5′-phosphate termini in ______-stranded DNA

A

DNA Ligases
Phosphodiester
Single
Double

55
Q

What is a recombinant DNA

A

artificial DNA made from combining DNA from multiple organisms.

56
Q

What is gene cloning?

A

The ability to isolate and make loads of identical copies of a fragment of DNA - ie a single gene

57
Q

What are vectors?

A

DNA molecule (often plasmid or virus) that is used as a vehicle to carry a particular DNA segment into a host cell as part of a cloning or recombinant DNA technique.

58
Q

What is the name of a DNA molecule (often plasmid or virus) that is used as a vehicle to carry a particular DNA segment into a host cell as part of a cloning or recombinant DNA technique.

A

Vector

59
Q

Define: Vector

A

A DNA molecule used to carry a DNA segment into a host cell

60
Q

What are the 5 steps to DNA recombination?

A
  1. isolate bulk DNA
  2. Isolate specific DNA sequence
  3. Ligation
  4. Transform bacteria (force it to take up vector)
  5. screen bacteria
61
Q
  1. isolate bulk DNA
  2. Isolate specific DNA sequence
  3. Ligation
  4. Transform bacteria (force it to take up vector)
  5. screen bacteria

Are the 5 steps for what process?

A

DNA recombination

62
Q

What are the 5 steps for DNA ecombinatin?

A
  1. isolate bulk DNA
  2. Isolate specific DNA sequence
  3. Ligation
  4. Transform bacteria (force it to take up vector)
  5. screen bacteria
63
Q

True or false: Viruses can be used as vectors

A

True

64
Q

YES/NO: is bacteriophage a vector?

A

Yes

65
Q

What is bacteriophage lambda?

A

A virus that infects bacteria

66
Q

Name a virus that infects bacteria

A

Bacterrophage lambda

67
Q

What is a plasmid?

A

A small circular DNA molecule found in bacteria and some other microscopic organisms

68
Q

A small circular DNA molecule found in bacteria and some other microscopic organisms

A

Plasmid

69
Q

What is dsDNA?

A

double stranded DNA

70
Q

What is a plasmid vector?

A

small extrachromosomal, circular DNA molecules which are replicated in appropriate bacterial hosts

71
Q

small extrachromosomal, circular DNA molecules which are replicated in appropriate bacterial hosts

A

plasmid vector

72
Q

True or false: Plasmids often carry antibiotic resistance genes

A

True

73
Q

What is multi cloning site?

A

Aka polylinker site: a short segment of DNA which contains many (up to ~20) restriction sites

74
Q

What is a polylinker site?

A

Aka multicloning site: a short segment of DNA which contains many (up to ~20) restriction sites

75
Q

What is a eukaryotic exon?

A

A protein coding sequence

76
Q

What is a eukaryotic intron?

A

an unexpressed or inoperative nucleotide sequence within a gene.

77
Q

Yes or no: Can mRNA be used as a template to make cDNA (complementary) for gene cloning?

A

yes

78
Q

a DNA polymerase enzyme that transcribes single-stranded RNA into DNA

A

a DNA polymerase enzyme that transcribes single-stranded RNA into DNA

79
Q

What is DNA polymerase?

A

(DNAP) is a type of enzyme that is responsible for forming new copies of DNA, in the form of nucleic acid molecules.

80
Q

What is an oligo (dT) Primer?

A

oligonucleotides that contain a segment of repeating deoxythymidines (dT)

81
Q

What is an oligoneucleotide?

A

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics.

82
Q

________________ are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics.

A

Oligonucleotides

83
Q

What are deoxythymidines (dT)?

A

the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA

84
Q

What is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA called

A

deoxythymidines (dT)

85
Q

What is poly (A)+

A

is the addition of a poly(A) tail to an RNA transcript

86
Q

Definition: is the addition of a poly(A) tail to an RNA transcript

A

Poly (A)+

87
Q

What is RNase H

A

Ribonucleases H are enzymes that cleave the RNA of RNA/DNA hybrids that form during replication and repair and which could lead to DNA instability if they were not processed.

88
Q

_____________ _ are enzymes that cleave the RNA of RNA/DNA hybrids that form during replication and repair and which could lead to DNA instability if they were not processed

A

Ribonucleases H

89
Q

Restriction Enzymes ___ DNA very specifically at _ to _bp

A

cut
4-8

90
Q

Restriction enzymes are homo_______ enzymes that recognize ___________ sequences.

A

homodimeric
palindromic

91
Q

Restriction enzymes leave ______ or _____ ends

A

Sticky
Blunt

92
Q

______ ends are useful for cloning vectors

A

sticky

93
Q

Vectors allow ___________ of gene fragmnents within host organism

A

propagation

94
Q

________ are bacterial vectors used frequently in gene cloning.

A

Plasmids

95
Q

Plasmids are ________ DNA molecules containing an origin of replication and __________ resistance gene.

A

circular
antibiotic

96
Q
A

Biochemistry BCC (7 decks)

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