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KEM350 > Chapter 4: Recombinant DNA techniques and molecular cloning > Flashcards

Chapter 4: Recombinant DNA techniques and molecular cloning Flashcards

1
Q

Nucleosides is combined with nitrogeneous bases, which gave two types, which?

A

Purines and pyrimidines

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2
Q

What is the bond called between nucleotides+

A

5’ - 3’ Phosphodiester bond

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3
Q

At which end does the phosphate bind the nuclesides? and which types of bond

A
  1. 5’

2. Ester bond

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4
Q

the sugar component in nuclesides in DNA is pentose, whats for RNA?

A

ribose

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5
Q

polynucleotide molecules are also called _____ that form macromolecular structures such as DNA and RNA

A

polymers

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6
Q

How many polynucleotide strand does DNA have ?

and what about the polarity?

A
  1. 2

2. oppposite direction polarity –> antiparallel structure

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7
Q

Which two purine bases do we have? and are they found both in DNA and RNA ?

A

Adenine (A) and Guanine (G)

yes

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8
Q

which one amoung the three pyrimidine bases is found both in DNA and RNA ?

A

Cytosine

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9
Q

Which 1/3 of the pyrimidine bases is only found for DNA ?

and for RNA ?

A

Thymine

Uracil

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10
Q

it is the sequence of ____ along a molecule that distinguishes one DNA (or RNA) from another

A

bases

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11
Q

Why is there no point in indicating the

sugar or phosphate groups?

A

since these are identical throughout the length of the

molecule

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12
Q

Adenine always pair with ___ for DNA
___ for RNA

Guanine always pair with ___

A

Thymine
Uracil
Cytosine

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13
Q

The major distinguishing feature of B-DNA is that it has approximately __ bases for
one turn of the double helix

A

10

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14
Q

In the B form of DNA, the major groove is ___ than the
minor groove –> DNA-binding proteins such as transcription factors bind
to the DNA molecule at the ___ major groove

A

wider

wider

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15
Q

DNA may adopt a left-handed helical

structure termed _-DNA

A

Z

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16
Q

The various forms of DNA serve to show that it
is not a static molecule, but dynamic and constantly in fl ux, and may be:
1. ___
2. ___
3. ___
at certain times.

A

coiled
bent
distorted

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17
Q

Although RNA almost always exists as a __ ___,
it often contains sequences within the same strand that are self-complementary, and
which can therefore base-pair if brought together by suitable ____ of the molecule.
A notable example is _____ which folds up to give a clover-leaf secondary
structure ( Figure 4.6 ).

A

single strand
folding
rRNA

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18
Q

The two anti-parallel strands of DNA are held together partly by the weak forces of
____ bonding between complementary bases and partly by _____ interactions
between adjacent, stacked base pairs, termed base-stacking.

A

hydrogen

hydrophobic

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19
Q

Little energy is required to seperate few base pairs –> giving few stretched of the DNA to open up to a single starnd but due to the fact that DNA usualy is in a ___ ___ then these streches will ___ again so the molecule as a whole will remin double stranded

If, however, a DNA solution is heated to approximately ___ or above, there will be enough kinetic energy to ___ the DNA completely, causing it to separate into single
strands, this can be followed spectrophotometrically
by monitoring the absorbance of light at ____

A 
biological temp
pair up
90 °C
denature
260 nm
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20
Q

Explain the hyperchromic effect

A

When DNA is seperated to single strands, spectrophotometric techniques can easier detect the absorbance at 260 nm compared to par wise stacked bases.

the effect is a phenomenon of the increased absorbance as DNA is denatired.

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21
Q

The absorbance at 260 nm may be plotted against the temperature of a DNA solution,
which will indicate that little denaturation occurs below approximately ___

A

70°C

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22
Q

The temperature at which 50% of the DNA is melted is termed the ___ ____

A

melting temperature ( T m )

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23
Q

If several different samples of DNA are melted, it is found that the Tm is highest for
those DNA molecules that contain the highest proportion of ____ and ____

A

cytosine (pyrimidine base)

guanine (purine base)

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24
Q

Tm can actually be used to estimate the percentage of __ and __ in a DNA sample

Why?

A

C
G

C and C form three hydrogen bonds when base-paired, whereas thymine and adenine form
only two. Because of the differential numbers of hydrogen bonds between A–T and
C–G pairs those sequences with a predominance of C–G pairs will require greater
energy to separate or denature them. The conditions required to separate a particular
nucleotide sequence are also dependent on environmental conditions such as salt
concentration.

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25
Q

Describe denaturation

A

If melted DNA is cooled, it is possible for the separated strands to reassociate only if the bases on both polynucletide strand can stack.

Which is a unlikely event due to the length of the DNA having many different genes.

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26
Q

Measurements of the rate of renaturation

can give information about the complexity of a DNA _____

A

preparation.

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27
Q

Strands of RNA and DNA will associate with each other, if their sequences are complementary –> double-stranded, hybrid molecules.

Similarly, strands of radioactively labelled RNA or DNA, when added to a denatured DNA preparation, will act as ___ for DNA molecules to which they are complementary. This hybridisation of complementary strands of nucleic acids is very useful for ____ a specific fragment of DNA from a complex mixture

A

probes

isolating

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28
Q 
It is also possible for small single-stranded fragments
of DNA (up to 40 bases in length), termed \_\_\_\_\_\_ , to hybridise to a denatured sample of DNA.

This type of hybridisation is termed ____ and again
is dependent on the base sequence of the oligonucleotide and the salt concentration
of the sample.

A

oligonucleotides

annealing

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29
Q

Each region of DNA that codes for a single RNA or protein is called a ___

A

gene

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30
Q

The entire set of genes in a virus, organelle or cell is termed ___

A

genome

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31
Q

Genomic DNA in nearly all procariotes and eucariotes are complexed woth proteins –> ____ DNA

A

chromosomal

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32
Q

Each gene is located at a particulat site which is termed ___

A

locus

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33
Q

The form of the gene is termed ___

A

allele

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34
Q

In mammalian DNA, each gene is present in two allelic forms, which two?

A

Homozygous – where all allele are identical

Heterozygous – where the allele’s may vary

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35
Q

The occurrence of different alleles at the same site in the genome is termed ___

A

polymorphism

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36
Q

In general, the more complex an organism the larger its genome, although this is not always the case, since many higher organisms have non-coding sequences, some of which are repeated numerous times and termed _____ DNA

For mammalian DNA we have two sub categories, which two ?

These two sub-categorical repetetive DNA sequences may be defined into so-called (3)!

microsatellite DNA is mainly composed of
dinucleotide repeats. These sequences are termed _____

A

repetitive

low-copy-number
& high-copy-number DNA (that
are dispersed throughout the genome)

classical satellite DNA
minisatellite
microsatellite DNA

polymorphic, which collectively termed
polymorphisms –> and vary between individuals; they also form the basis of genetic
fi ngerprinting.

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37
Q

Which source of polymorphic diversity is known to be present in genomes?

A

SNPs

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38
Q

What are SNPs?

A

substitutions of one base at a precise location within the genome

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39
Q

Those SNPs that are present in coding regions are termed

A

cSNPs

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40
Q 
Interest in SNPs lies in the fact that
these polymorphisms may account for the differences in 
1.
2.
3.
A

disease susceptibility
drug metabolism
response to environmental factors between individuals

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41
Q

Which association can tag SNPs provide

A

association between the haplotype and a disease

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42
Q

Explain metaphase for higher organisms

A

is a particular point in the cell divition cycle which can be used to indetifie higher organims based on the shape and size of the genetic material

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43
Q

at the point of the metaphase what happens with DNA ?

A

it condenses and give a number of very distict chromosome structures having different morphological characteristics that can be studies by staing and subsequent analysis with light microscopy

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44
Q

explain the term karyotype!

A

is aterm used for the array of all chromosomes in a organism

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45
Q

with the ___ and the ____ one may derive an indicator of an disorder based on the number, shape and size of chromosome strucures in a organism

A

karyotype

metaphase

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46
Q

what does the rate of renaturation give valuable information of?

A

genomic complexity (measured in base-pairs)

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47
Q

3 preparative steps in studying the renaturation kinetics, which three?

A
  1. cutting DNA into 1kb length fragments
  2. Complete denaturing by heating above Tm
  3. renaturation by 10 degrees below Tm, while monitoring
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48
Q

Two ways to monitor the renaturation

A
  1. decrease in absorbance (260 nm, hypochromic effect)

2. passsing sampls at intervals through a hydroxylapatite column

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49
Q

What will the degree of renaturation depend on?

A

conc of double stranded DNA prior to denaturation
t
duration of renaturation in seconds

50
Q

DNA encodes the primary sequence of a protein by utilising sets of three ____,
termed a codon or triplet, to encode a particular __ __

A

nucleotides

amino acid

51
Q

The four bases (A, C, G and T) present in DNA allow a possible __ triplet combinations;
however, since there are only 20 naturally occurring amino acids, more than one codon may encode an
amino acid. This phenomenon is termed the _____

A

64

degeneracy of the genetic code

52
Q

Only one start codon exists (___), which also codes
for the amino acid methionine, whereas three dedicated stop codons are available
(___, ___ and ___)

A

ATG

TAT, TAG and TGA

53
Q

Describe the term open reading frame (ORF)

A

sequence fl anked by a start and a stop codon
containing a number of codons that may be read in-frame to represent a continuous
protein sequence

54
Q

Describe DNA methylation!

catalyzed by!

.% of human DNA is methylated –> epigenome

further modified by Tet enzymes that oxidate 5mC –> 5hmC, as a demthylation process

A

modification by adding a methyl group to 5’ carbon on cytosine (C-phosphate-G) –> methylcytosine (5mC) –> fifth base in DNA

methyltransferase

1.5

55
Q

high CpG density regions occur at __% of gene promoter regions and methylation
of these sites inhibits gene expression

A

40

56
Q

In addition to DNA methylation, we also have a modification that targets gene expression, tell me how!

A

modifi cation of histone proteins and some small RNA molecules inv

57
Q

In general, DNA in
eukaryotic cells is confined to the ___ and ___ such as mitochondria or
chloroplasts, which contain their own genome

A

nucleus

organelles

58
Q

The predominant RNA species are, however, normally located within the ___ and fall into three classes

A

cytoplasm

59
Q

DNA in:
Prokaryotic cells associated with nucleoid proteins by ____

eukaryotic cells have
many levels of packaging of the DNA within the ____, involving a variety of DNA
binding proteins.

A

supercoiling (histones –> nucleosome–> chromatin)

nucleus

60
Q

Which process is carried out by the enzyme DNA helicase?

A

unwinding of DNA

61
Q

in order to prevent

the single strands from reannealing, small proteins termed ____ attach to each single DNA strand

A

SSBs

62
Q

__ _____ __ also employs the original DNA as a template for synthesis of a DNA strand, using the RNA primer as a starting point

A

DNA polymerase III (DNA pol III)

63
Q

The primer is vital since it leaves an exposed __ - ___ __. This is necessary since DNA pol III can only add new nucleotides to the 3’
end

A

3-hydroxyl

group

64
Q

Synthesis of the DNA strand occurs only in a _to _ direction from the RNA primer. This DNA strand is usually termed the __ ___ and provides the means for continuous DNA synthesis.

A

5
3
leading strand

65
Q

the lagging strand is formed by ___ fragemnts

A

Okazaki

66
Q

Explain semi-conservative replication

A

The
net result of the replication is that the original DNA is replaced by two molecules,
each containing one ‘old’ and one ‘new’ strand

67
Q

bakterial protection by differential metylation of host DNA with ___ and ___

A

DNA methylase and restriction endonuclease

68
Q

The recombination–repair systems in eukaryotic cells share some common features
with prokaryotes, although the mechanisms in ____ cells are more complex.

A

eukaryotic

69
Q

In eukaryotes, __ different R NA polymerases exist

A

three

70
Q

In procariotes, promoters comprimise __ highly conserved sequence elements, named!

A

2

TATA box (-10) - involved in formation of transcription initiation complex

‘GC-rich’ sequence (-25) important to bind RNA polymerase. HOUSEKEEPING GENES

71
Q

Gene regulation occurs in most cases at the
level of ____, and primarily by the rate of ___ ___, although
control may also be by modulation of mRNA stability, or at other levels such as translation

A

transcription

transcription initiation

72
Q

Terminator sequences are less well characterised, but are thought to involve
nucleotide sequences near the end of mRNA with the capacity to form a __ ____,
followed by a run of _ residues, which may constitute a termination signal for RNA
polymerase.

A

hairpin loop

U

73
Q

CAT box is important deteminant for ___ efficienty

A

promoter

74
Q

Prokaryotic gene organisation differs from that found in eukaryotes in a number of
ways. Prokaryotic genes are generally found as ____ coding sequences that
are not interrupted. Moreover, they are frequently found clustered into ___ that
contain genes that relate to a particular function

A

continuous

operons

75
Q

Post-transcriptional gene regulation and its control its complex. One important control mechanism is through ___
These are similar to siRNAs found in the RNA interference

A

miRNA by specific binding to 3’ untranslated regions in mRNA

76
Q

Transcription of a eukaryotic gene results in the production of a hnRNA

A

hnRNA

77
Q

Transcription of a eukaryotic gene results in the production of a hnRNA

  1. m7Gppp -5’ - … - 3’ (hnRNA)-
    poly(A)
    tail
A

hnRNA

78
Q

Unlike prokaryotic transcripts, those from eukaryotes have their coding sequence
(expressed regions or ____ ) interrupted by non-coding sequences

A

exons

79
Q

The process by intron solicing is mediated by?

and intron-exon boundaries are remove __-__ sequences before mature mRNA can be formed

A

snRNAs found in splicesome

GU AG

80
Q

Messenger RNA molecules are read and translated into proteins by complex RNA–
protein particles termed ____

A

ribosomes

81
Q

In prokaryotic cells the ribosome binds to the 5 end of the mRNA at a sequence
known as a ribosome binding site or sometimes termed the ___-___sequence

In eukaryotes, the situation is similar, but involves a ___
sequence (named after Marylin Kozak) located around the initiation codon

A

Shine-Dalgarno

Kozak

82
Q

linking an amino acid to its specific tRNA is termed charging and is carried out by
the enzyme aminoacyl tRNA _____.

A

synthetase

83
Q

ribosome
allows the formation of a peptide bond between the amino acids. The process is also
under the control of an enzyme, ___ transferase.

A

peptidyl

84
Q

Control of Protein Production: RNA Interference:

siRNA (dicer on dsRNA) control the the avaliablity for mRNA to be ___ with RNA-inducer silencing complex

A

translated

85
Q

Ideally,
cell walls, if present, should be digested ____ (e.g. lysozyme treatment of
bacteria), and the cell membrane should be ____ using detergent.

A

enzymatically

solubilized

86
Q

DNA is recovered from cells by cell rupture to prevent DNA from ___ by mechanical schearing.

The extraction process (4*c) usually done in ___, then

  1. cell wall is digested ___
  2. Cell membrane solubilized using ___

After relase of nucleic acid, RNA can be removed by __

and other contaminants can be removed by ___ saturated phenol or phenol/chloroform mix

the solution now have a organic phase when centrifuted (protein content) at bottom, which we dont want

Finally, the DNA preparation is mixed with two volumes
of absolute ___, and the DNA allowed to precipitate out of solution in a
freezer.

After centrifugation, the ___ pellet is redissolved in a buffer containing EDTA
to inactivate any DNases present. This solution can be stored at 4 °C for at least a month.

A

fragmenting

EDTA to chelate mg2+ ions used by enzymes to degrade DNA.

enzymatically

Detergent

heat-treated (rapid rematuration upon cooling) RNase

water

ethanol

DNA

87
Q

If the DNA from a specific organelle or viral particle is needed, should extraction be done right away?

A

no, use DG ultracentrifugation through caesium chloride

88
Q

How can conc. of DNA be determined?

and how are contaminant identified?

what ratio suggests sample free from contaminants?

A

measuring absorbance at 260 nm in a spectrophotometer and use equation (p.98)

UV spectroscopy from 200 to 300 nm

1.8 (ration between absorbances 260 & 280)

89
Q

Regarding SDS-PAGE, after separation followed by staining having dyes that bind to DNA by:

A

insertion between stacked base pairs (intercalation) and exhibit a strong green with SYBRgreen under UV.

90
Q

Agarose
gels can be used to separate molecules larger than about ___ bp. For higher resolution
or for the effective separation of shorter DNA molecules, ___ gels are the
preferred method.

A

100

polyacrylamide

91
Q

The DNA may be recovered from the gel fragment in various ways from electrophoresis experiment., how

A
  1. glass rod + agarase

2. electroelution

92
Q

Explain Restriction mapping!

A

size analysis of restricion fragments of DNA by double digestion but bioinformatic analysis is needed to contruct a map becuase sites can be close to each other.

93
Q

Southern blotting is a ___ blotting method

What does it provide?

A

nucleic acid

indication of a specific fragments by DNA from gel (soacked in alkali) to nitrocelluse or nylon membrane the followed with electro-tratement or buffer drawing for capillary actionthen washing

94
Q

The same basic process of nucleic acid blotting can be used to transfer RNA from gels
onto similar membranes. This allows the identification of specific mRNA sequences of
a defi ned length by hybridization to a labeled gene probe and is known as
___ ____

a special version where one use nylon membrane and directly analyse without gel electrophoresis is called ___

A

Northern blotting

slot

95
Q

A gene probe can be!

how is it commonly produced!

A

dye for example.

by artificial chemical synthesis.

96
Q

what are phosphoramiditis?

A

monomers that are modified dNTPs

, which via artificial chemical synthesis are linked together via 5’ carbon, and

97
Q

Why is there no need to synthesis oligonucleotides with more than 20 amino acids?

A

a) since this should hybridise
efficiently with any complementary
sequences
b) should be specific for one gene.

Ideally, a section of the protein should be chosen that contains as many tryptophan
and methionine residues as possible, since these have unique codons, and there will
therefore be fewer possible base sequences that could code for that part of the protein.

98
Q

What is the most common radioactive label?

A

phosphorous-32

99
Q

Describe direct labelling

is it more popular?

A

allows an ezyme reporter to be coupled directly to the DNA

no

100
Q

What is the simplest form of labeling DNA?

A

5’ or 3’ end labelling

101
Q

5’ end labbeling where the phosphate on the N termal 5’ group is replaces with a labelled phosfate via 2 enzymes, which two?

A

alkaline phosphotase

polynucleotide kinase

102
Q

With 3’ end labbeling a labelled dNTP is added by terminal transferase, what happens with the DNA sequence?

A

its altered, thus may effect hybridization to its target sequence

103
Q

3’ end labbeling probes are said to have ____ specific activity in comparison to probes that incorporate labels along the length of the DNA.

A

lower

104
Q

Why do we use PCR?

A

in order to amplify a gene, analogeous to DNA replication process but artifical

105
Q

What are the stages in PCR?

A

Denaturation
Annealing
extension

106
Q

In PCR what happens in the annealing step?

A

the hybridization of the two oligonucleotide sequenses which acts as primers due to their free 3’-hydroxyl group for DNA polymerase

107
Q

the extension step in PCR is carried out by which thermostable DNA polymerase?

A

Taq DNA polymerase

108
Q

What is the biggest senitivity related to the drawback regarding PCR?

A

contamination

109
Q

How can the contamination aspect of PCR bet tackaled (two ways)?

A

by UV irradiation to damge already amplified products so they cant be used as templates

incorporate uracil into the PCR and then treat the products with enzyme UNG

110
Q

Following the isolation and purifi cation of genomic DNA it is possible to specifi cally
fragment it with enzymes termed restriction ____

A

endonucleases

111
Q

It is important to note that every copy of a given DNA molecule from a
specifi c organism will yield the ____ set of fragments when digested with a particular
enzyme

A

same

112
Q

the process of ligation is ___ dependent when making the covalent bond between 5’ end to 3’ end of two fragments. Often carried out at __ degrees celcius

A

ATP

10

113
Q

For the cloning of any molecule of DNA it is necessary for that DNA to be incorporated
into a cloning vector becuase it has ____ functions

A

replication

114
Q

cloning vectors have in general been developed from naturally ocurring molecules, such as ___ plasmids and bac___ or cosmids

A

bacterial

bacteriophages

115
Q

Many bacteria contain an extrachromosomal element of DNA, termed a ___,
which is a relatively small, covalently closed circular molecule, carrying genes for
antibiotic resistance, conjugation or the metabolism of ‘unusual’ substrates –> thus excellent as vectors —modification—> cloning vectors (has better transformation than natural plasmids)

A

plasmid

116
Q

Insertional inactivation is a useful selection method for:

A

identifying recombinant vectors with inserts, where cells containing the genes of resistance to antibiotics will not survive on a medium when a restriction enzyme attacks the restriction site inside antiobiotic resistance gene.

117
Q

E-coli does not normally transform ligated DNA into cells but can be induced by prior treatment with:

When the E-coli cells are transformed what term is used to tell this?

How do we ensure that the ligated DNA is taken up?

A

ca2+ at 4 degrees celcius

competent

heat shock

118
Q

If the digested plasmids are treated
with the enzyme ____ ____ prior to ligation, re-circularisation will be prevented,
since this enzyme removes the 5-phosphate groups that are essential for ligation

A

alkaline phosphatase

119
Q

In order for a gene insert into a bakterial cell regarding transcription it must have particular ___ sequences

and it must contain a ____-binding-site , placed just before the coding region.

these sequences are before the restriction sites, what are they called?

A

promoter
ribosome
regulatory sequences forming opreon

120
Q

Since the mRNA produced from the gene is read as triplet codons, the
inserted sequence must be placed so that its reading frame is in phase with the ___
sequence.

A

regulatory

121
Q

A non-PCR-based method that is traditionally used is

A

northern blotting: for detection
of particular RNA transcripts by blotting extracted mRNA and immobilising it on a
nylon membrane with subsequent hybridization with labelled gene probes

or

122
Q

reporter genes, such as those using

green fluorescent protein ( GFP) allow quantification of gene transcription in response to ____ activators.

A

transcriptional

KEM350 (13 decks)

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