Recombinant DNA Technoloy 1 Flashcards
1
Q
What is Histone?
A
A protein that provides structural support for a chromosome.
2
Q
A protein that provides structural support for a chromosome.
A
Histone
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.
4
Q
What is genome sequencing?
A
a comprehensive list of every base pair sequence of DNA.
5
Q
what is homogenisation?
A
A process that involves breaking apart cells - releasing organelles and cytoplasm.
6
Q
A process that involves breaking apart cells - releasing organelles and cytoplasm.
A
Homogenisation
7
Q
From the Greek root…… means “of the same kind.
A
homogenes
8
Q
How is DNA prepared for (?)
A
- Get my sample from the animal
- homogenise the cells or tissues
- use classic phenol extraction/centrifugation
- the sample will separate into an aqueous layer and phenol layer.
- the aqueous layer will contain RNA nad DNA ad the phenol layer will contain protein.
6 - Ethanol precipitation - purifying or concentrating DNA, RNA, Polysaccharides.
9
Q
What is DNA recombination?
A
The process during which a stretch of DNA recombines and produces a new allelic combination
10
Q
what is phenol extraction?
A
Separating nucleic acids from other cellular substances
11
Q
what is the process of Separating nucleic acids from other cellular substances called?
A
Phenol Extraction
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.
13
Q
How many layers are there in the sample after a phenol extraction/cetrifugation? and what are they?
A
Aqueous Layer
phenol layer
14
Q
What substance is contained in an aqueous layer after phenol extraction/centrifugation?
A
DNA + RNA
15
Q
What substance is contained in a phenol layer after phenol extraction/centrifugation?
A
Proteins
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.
17
Q
Why is ethanol used on DNA extraction?
A
Ethanol precipitation is often used to obtain DNA molecules extruded from cells.
18
Q
_________ __________ is often used to obtain DNA molecules extruded (thrust, forced out) from cells.
A
Ethanol precipitation
19
Q
Name 3 uses of Type II Restriction Endonucleases
A
- Gene cloning
- DNA fragmentation
- DNA analysis
20
Q
What is DNA fragmentation?
A
The separation or breaking of DNA strands into pieces
21
Q
Gene cloning, DNA fragmentation and DNA analysis are three ways in which ___ __ ___________ _____________ can be usedin DNA extraction.
A
Type II Restriction Endonucleases
22
Q
Define: nuclease
A
An enzyme that cleaves the chains of nucleotides in nucleic acids into smaller units.
23
Q
How many types of Type II Restriction Endonucleases are recognised?
A
over 3500
24
Q
How many different DNA sequences do the 3500+ Type II enzymes recognise?
A
350+
25
Why are type I restriction enzymes preferable to type II?
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..
26
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.
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..
27
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..
True
28
True or false:
Type II restriction enzymes will randomly cut DNA, at times far from the recognition site.
False
29
True or false:
Type II will cut DNA from the recognition site at the defined position..
True
30
What is EcoRI?
A restriction endonuclease enzyme isolated from species E. coli
31
A restriction endonuclease enzyme isolated from species E. coli
What is EcoRI.
32
What does EcoR1 do in genetic engineering?
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.
33
What is a palindrome?
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'
34
A word, phrase, or sequence that reads the same backwards as forwards (An anti parallel DNA sequence)
Palindrome
35
What are the two manners of cut to target DNA that are useful in genetic engineering?
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.
36
What is the difference between a sticky end cut and a blunt end cut?
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.
37
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.
Sticky End
38
A _____ ___ cut leave no exposed unpaired bases.
Blunt end
39
Why is it called a sticky end cut?
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
This orthodox Type IIP enzyme, binds to DNA in the absence of metal ions and cleaves the DNA sequence 5′-GGTAC^C-3
Kpn1
41
True of false: A Kpn1 enzyme cut produces a blunt end cut with no overhangs
False.
42
True of false: A Kpn1 enzyme cut produces sticky end cut with overhang
True
43
True of false: An EcoRV enzyme cut produces sticky end cut with overhang
False
44
True of false: A, EcoRV enzyme cut produces a blunt end cut with no overhangs
True
45
How often will recombination endonucleases cut?
every 44 (256) bases, while a 6 bp cutter cleaves every 46 (4096) bases.
46
True of false:
Restriction enzymes with shorter recognition sequences cut more frequently than those with longer recognition sequences
True
47
In the digestion frequency ofrecombination endonucleases, what does 4n represent?
The number of bases G/A/C/T
48
In the digestion frequency of recombination endonucleases, what does 'n' represent?
The length of recognition sequence.
49
How many enzyme cuts are made in IN n=4?
256 bp (base pairs)
50
How many enzyme cuts are made in IN n=6?
4096 bp (base pairs)
51
What is DNA ligation?
Joining of DNA strands together by catalysing the formation of a phosphodiester bond
52
Name the 3 steps of DNA ligation
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
Molecular basis of DNA ligation
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
___ _______ catalyse the formation of _____________ bonds at ______-strand breaks between adjacent 3′-hydroxyl and 5′-phosphate termini in ______-stranded DNA
DNA Ligases
Phosphodiester
Single
Double
55
What is a recombinant DNA
artificial DNA made from combining DNA from multiple organisms.
56
What is gene cloning?
The ability to isolate and make loads of identical copies of a fragment of DNA - ie a single gene
57
What are vectors?
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
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.
Vector
59
Define: Vector
A DNA molecule used to carry a DNA segment into a host cell
60
What are the 5 steps to DNA recombination?
1. isolate bulk DNA
2. Isolate specific DNA sequence
3. Ligation
4. Transform bacteria (force it to take up vector)
5. screen bacteria
61
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?
DNA recombination
62
What are the 5 steps for DNA ecombinatin?
1. isolate bulk DNA
2. Isolate specific DNA sequence
3. Ligation
4. Transform bacteria (force it to take up vector)
5. screen bacteria
63
True or false: Viruses can be used as vectors
True
64
YES/NO: is bacteriophage a vector?
Yes
65
What is bacteriophage lambda?
A virus that infects bacteria
66
Name a virus that infects bacteria
Bacterrophage lambda
67
What is a plasmid?
A small circular DNA molecule found in bacteria and some other microscopic organisms
68
A small circular DNA molecule found in bacteria and some other microscopic organisms
Plasmid
69
What is dsDNA?
double stranded DNA
70
What is a plasmid vector?
small extrachromosomal, circular DNA molecules which are replicated in appropriate bacterial hosts
71
small extrachromosomal, circular DNA molecules which are replicated in appropriate bacterial hosts
plasmid vector
72
True or false: Plasmids often carry antibiotic resistance genes
True
73
What is multi cloning site?
Aka polylinker site: a short segment of DNA which contains many (up to ~20) restriction sites
74
What is a polylinker site?
Aka multicloning site: a short segment of DNA which contains many (up to ~20) restriction sites
75
What is a eukaryotic exon?
A protein coding sequence
76
What is a eukaryotic intron?
an unexpressed or inoperative nucleotide sequence within a gene.
77
Yes or no: Can mRNA be used as a template to make cDNA (complementary) for gene cloning?
yes
78
a DNA polymerase enzyme that transcribes single-stranded RNA into DNA
a DNA polymerase enzyme that transcribes single-stranded RNA into DNA
79
What is DNA polymerase?
(DNAP) is a type of enzyme that is responsible for forming new copies of DNA, in the form of nucleic acid molecules.
80
What is an oligo (dT) Primer?
oligonucleotides that contain a segment of repeating deoxythymidines (dT)
81
What is an oligoneucleotide?
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics.
82
________________ are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics.
Oligonucleotides
83
What are deoxythymidines (dT)?
the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA
84
What is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA called
deoxythymidines (dT)
85
What is poly (A)+
is the addition of a poly(A) tail to an RNA transcript
86
Definition: is the addition of a poly(A) tail to an RNA transcript
Poly (A)+
87
What is RNase H
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
_____________ _ 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
Ribonucleases H
89
Restriction Enzymes ___ DNA very specifically at _ to _bp
cut
4-8
90
Restriction enzymes are homo_______ enzymes that recognize ___________ sequences.
homodimeric
palindromic
91
Restriction enzymes leave ______ or _____ ends
Sticky
Blunt
92
______ ends are useful for cloning vectors
sticky
93
Vectors allow ___________ of gene fragmnents within host organism
propagation
94
________ are bacterial vectors used frequently in gene cloning.
Plasmids
95
Plasmids are ________ DNA molecules containing an origin of replication and __________ resistance gene.
circular
antibiotic
96
