Chapter 4: Recombinant DNA techniques and molecular cloning Flashcards
Nucleosides is combined with nitrogeneous bases, which gave two types, which?
Purines and pyrimidines
What is the bond called between nucleotides+
5’ - 3’ Phosphodiester bond
At which end does the phosphate bind the nuclesides? and which types of bond
- 5’
2. Ester bond
the sugar component in nuclesides in DNA is pentose, whats for RNA?
ribose
polynucleotide molecules are also called _____ that form macromolecular structures such as DNA and RNA
polymers
How many polynucleotide strand does DNA have ?
and what about the polarity?
- 2
2. oppposite direction polarity –> antiparallel structure
Which two purine bases do we have? and are they found both in DNA and RNA ?
Adenine (A) and Guanine (G)
yes
which one amoung the three pyrimidine bases is found both in DNA and RNA ?
Cytosine
Which 1/3 of the pyrimidine bases is only found for DNA ?
and for RNA ?
Thymine
Uracil
it is the sequence of ____ along a molecule that distinguishes one DNA (or RNA) from another
bases
Why is there no point in indicating the
sugar or phosphate groups?
since these are identical throughout the length of the
molecule
Adenine always pair with ___ for DNA
___ for RNA
Guanine always pair with ___
Thymine
Uracil
Cytosine
The major distinguishing feature of B-DNA is that it has approximately __ bases for
one turn of the double helix
10
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
wider
wider
DNA may adopt a left-handed helical
structure termed _-DNA
Z
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.
coiled
bent
distorted
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 ).
single strand
folding
rRNA
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.
hydrogen
hydrophobic
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 ____
biological temp pair up 90 °C denature 260 nm
Explain the hyperchromic effect
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.
The absorbance at 260 nm may be plotted against the temperature of a DNA solution,
which will indicate that little denaturation occurs below approximately ___
70°C
The temperature at which 50% of the DNA is melted is termed the ___ ____
melting temperature ( T m )
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 ____
cytosine (pyrimidine base)
guanine (purine base)
Tm can actually be used to estimate the percentage of __ and __ in a DNA sample
Why?
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.
Describe denaturation
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.
Measurements of the rate of renaturation
can give information about the complexity of a DNA _____
preparation.
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
probes
isolating
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.
oligonucleotides
annealing
Each region of DNA that codes for a single RNA or protein is called a ___
gene
The entire set of genes in a virus, organelle or cell is termed ___
genome
Genomic DNA in nearly all procariotes and eucariotes are complexed woth proteins –> ____ DNA
chromosomal
Each gene is located at a particulat site which is termed ___
locus
The form of the gene is termed ___
allele
In mammalian DNA, each gene is present in two allelic forms, which two?
Homozygous – where all allele are identical
Heterozygous – where the allele’s may vary
The occurrence of different alleles at the same site in the genome is termed ___
polymorphism
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 _____
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.
Which source of polymorphic diversity is known to be present in genomes?
SNPs
What are SNPs?
substitutions of one base at a precise location within the genome
Those SNPs that are present in coding regions are termed
cSNPs
Interest in SNPs lies in the fact that these polymorphisms may account for the differences in 1. 2. 3.
disease susceptibility
drug metabolism
response to environmental factors between individuals
Which association can tag SNPs provide
association between the haplotype and a disease
Explain metaphase for higher organisms
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
at the point of the metaphase what happens with DNA ?
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
explain the term karyotype!
is aterm used for the array of all chromosomes in a organism
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
karyotype
metaphase
what does the rate of renaturation give valuable information of?
genomic complexity (measured in base-pairs)
3 preparative steps in studying the renaturation kinetics, which three?
- cutting DNA into 1kb length fragments
- Complete denaturing by heating above Tm
- renaturation by 10 degrees below Tm, while monitoring
Two ways to monitor the renaturation
- decrease in absorbance (260 nm, hypochromic effect)
2. passsing sampls at intervals through a hydroxylapatite column
What will the degree of renaturation depend on?
conc of double stranded DNA prior to denaturation
t
duration of renaturation in seconds
DNA encodes the primary sequence of a protein by utilising sets of three ____,
termed a codon or triplet, to encode a particular __ __
nucleotides
amino acid
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 _____
64
degeneracy of the genetic code
Only one start codon exists (___), which also codes
for the amino acid methionine, whereas three dedicated stop codons are available
(___, ___ and ___)
ATG
TAT, TAG and TGA
Describe the term open reading frame (ORF)
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
Describe DNA methylation!
catalyzed by!
.% of human DNA is methylated –> epigenome
further modified by Tet enzymes that oxidate 5mC –> 5hmC, as a demthylation process
modification by adding a methyl group to 5’ carbon on cytosine (C-phosphate-G) –> methylcytosine (5mC) –> fifth base in DNA
methyltransferase
1.5
high CpG density regions occur at __% of gene promoter regions and methylation
of these sites inhibits gene expression
40
In addition to DNA methylation, we also have a modification that targets gene expression, tell me how!
modifi cation of histone proteins and some small RNA molecules inv
In general, DNA in
eukaryotic cells is confined to the ___ and ___ such as mitochondria or
chloroplasts, which contain their own genome
nucleus
organelles
The predominant RNA species are, however, normally located within the ___ and fall into three classes
cytoplasm
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.
supercoiling (histones –> nucleosome–> chromatin)
nucleus
Which process is carried out by the enzyme DNA helicase?
unwinding of DNA
in order to prevent
the single strands from reannealing, small proteins termed ____ attach to each single DNA strand
SSBs
__ _____ __ also employs the original DNA as a template for synthesis of a DNA strand, using the RNA primer as a starting point
DNA polymerase III (DNA pol III)
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
3-hydroxyl
group
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.
5
3
leading strand
the lagging strand is formed by ___ fragemnts
Okazaki
Explain semi-conservative replication
The
net result of the replication is that the original DNA is replaced by two molecules,
each containing one ‘old’ and one ‘new’ strand
bakterial protection by differential metylation of host DNA with ___ and ___
DNA methylase and restriction endonuclease
The recombination–repair systems in eukaryotic cells share some common features
with prokaryotes, although the mechanisms in ____ cells are more complex.
eukaryotic
In eukaryotes, __ different R NA polymerases exist
three
In procariotes, promoters comprimise __ highly conserved sequence elements, named!
2
TATA box (-10) - involved in formation of transcription initiation complex
‘GC-rich’ sequence (-25) important to bind RNA polymerase. HOUSEKEEPING GENES
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
transcription
transcription initiation
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.
hairpin loop
U
CAT box is important deteminant for ___ efficienty
promoter
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
continuous
operons
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
miRNA by specific binding to 3’ untranslated regions in mRNA
Transcription of a eukaryotic gene results in the production of a hnRNA
hnRNA
Transcription of a eukaryotic gene results in the production of a hnRNA
- m7Gppp -5’ - … - 3’ (hnRNA)-
poly(A)
tail
hnRNA
Unlike prokaryotic transcripts, those from eukaryotes have their coding sequence
(expressed regions or ____ ) interrupted by non-coding sequences
exons
The process by intron solicing is mediated by?
and intron-exon boundaries are remove __-__ sequences before mature mRNA can be formed
snRNAs found in splicesome
GU AG
Messenger RNA molecules are read and translated into proteins by complex RNA–
protein particles termed ____
ribosomes
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
Shine-Dalgarno
Kozak
linking an amino acid to its specific tRNA is termed charging and is carried out by
the enzyme aminoacyl tRNA _____.
synthetase
ribosome
allows the formation of a peptide bond between the amino acids. The process is also
under the control of an enzyme, ___ transferase.
peptidyl
Control of Protein Production: RNA Interference:
siRNA (dicer on dsRNA) control the the avaliablity for mRNA to be ___ with RNA-inducer silencing complex
translated
Ideally,
cell walls, if present, should be digested ____ (e.g. lysozyme treatment of
bacteria), and the cell membrane should be ____ using detergent.
enzymatically
solubilized
DNA is recovered from cells by cell rupture to prevent DNA from ___ by mechanical schearing.
The extraction process (4*c) usually done in ___, then
- cell wall is digested ___
- 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.
fragmenting
EDTA to chelate mg2+ ions used by enzymes to degrade DNA.
enzymatically
Detergent
heat-treated (rapid rematuration upon cooling) RNase
water
ethanol
DNA
If the DNA from a specific organelle or viral particle is needed, should extraction be done right away?
no, use DG ultracentrifugation through caesium chloride
How can conc. of DNA be determined?
and how are contaminant identified?
what ratio suggests sample free from contaminants?
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)
Regarding SDS-PAGE, after separation followed by staining having dyes that bind to DNA by:
insertion between stacked base pairs (intercalation) and exhibit a strong green with SYBRgreen under UV.
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.
100
polyacrylamide
The DNA may be recovered from the gel fragment in various ways from electrophoresis experiment., how
- glass rod + agarase
2. electroelution
Explain Restriction mapping!
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.
Southern blotting is a ___ blotting method
What does it provide?
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
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 ___
Northern blotting
slot
A gene probe can be!
how is it commonly produced!
dye for example.
by artificial chemical synthesis.
what are phosphoramiditis?
monomers that are modified dNTPs
, which via artificial chemical synthesis are linked together via 5’ carbon, and
Why is there no need to synthesis oligonucleotides with more than 20 amino acids?
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.
What is the most common radioactive label?
phosphorous-32
Describe direct labelling
is it more popular?
allows an ezyme reporter to be coupled directly to the DNA
no
What is the simplest form of labeling DNA?
5’ or 3’ end labelling
5’ end labbeling where the phosphate on the N termal 5’ group is replaces with a labelled phosfate via 2 enzymes, which two?
alkaline phosphotase
polynucleotide kinase
With 3’ end labbeling a labelled dNTP is added by terminal transferase, what happens with the DNA sequence?
its altered, thus may effect hybridization to its target sequence
3’ end labbeling probes are said to have ____ specific activity in comparison to probes that incorporate labels along the length of the DNA.
lower
Why do we use PCR?
in order to amplify a gene, analogeous to DNA replication process but artifical
What are the stages in PCR?
Denaturation
Annealing
extension
In PCR what happens in the annealing step?
the hybridization of the two oligonucleotide sequenses which acts as primers due to their free 3’-hydroxyl group for DNA polymerase
the extension step in PCR is carried out by which thermostable DNA polymerase?
Taq DNA polymerase
What is the biggest senitivity related to the drawback regarding PCR?
contamination
How can the contamination aspect of PCR bet tackaled (two ways)?
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
Following the isolation and purifi cation of genomic DNA it is possible to specifi cally
fragment it with enzymes termed restriction ____
endonucleases
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
same
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
ATP
10
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
replication
cloning vectors have in general been developed from naturally ocurring molecules, such as ___ plasmids and bac___ or cosmids
bacterial
bacteriophages
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)
plasmid
Insertional inactivation is a useful selection method for:
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.
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?
ca2+ at 4 degrees celcius
competent
heat shock
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
alkaline phosphatase
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?
promoter
ribosome
regulatory sequences forming opreon
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.
regulatory
A non-PCR-based method that is traditionally used is
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
reporter genes, such as those using
green fluorescent protein ( GFP) allow quantification of gene transcription in response to ____ activators.
transcriptional
