4 | Recombinant DNA Techniques and Molecular Cloning Flashcards
What is nucleotide made up from?
nucleoside and phosphate group
What is a nucleoside made up from?
DNA: 2-deoxyribose + nitrogenous base
RNA: ribose + nitrogenous base
What bond is between adjacent sugars?
5’-to-3’ phosphodiester bond
Which two purine bases do we have?
A and G
Which 3 pyrimidine bases do we have?
C, U (only RNA) and T (only DNA)
what is the hyper chromic effect?
A phenomenon at which the absorbance at 260 nm increases as the DNA becomes denatured
The temperature at which 50% of the DNA is melted is termed the?
melting temperature
Measurements of the rate of renaturation
can give information about the complexity of a DNA preparation, how can it be so?
Because a stable double-stranded molecule will only be formed if the complementary strands collide in such a way that their bases are paired precisely, which is a higher probability in the event of reassociate for shorter DNAs
Each gene is located at a particular position along the chromosome, termed the ?
locus
particular form of the gene is termed the
allele
In mammalian DNA, each gene is present in two allelic forms that may be
identical (_____) or that may vary (____)
homozygous
heterozygous
The occurrence of different alleles at the same site in the genome is termed
polymorphism
What are SNPs?
SNPs are substitutions of one base at a precise location within the genome
What phase is resp. for DNA condensing to form a number of very distinct chromosome structures at a particular point in the cell division cycle.
And the complete array of chromosomes in an organism is termed
meta
Karyotype
A sequence flanked by a start (ATG) and a stop codon (TAT / TAG / TGA), containing a number of codons that may be read in-frame to represent a continuous
protein sequence is termed ?
ORF
Within epigenetic, DNA methylation is the most important modification, what’s happening?
addition of a methyl (CH 3 ) group to the 5 -carbon on cytosine to give methylcytosine ( 5mC ), catalysed by DNA methyltransferases. giving a CpG pattern.
What main 3 RNA types are there?
In eukaryotic cells alone a further group of RNA molecules termed small nuclear
RNA ( snRNA) is present, which functions within the nucleus and promotes the?
mRNA
rRNA
tRNA
maturation of mRNA molecules
micro-RNA and small interfering RNA are present and known to contribute to
modulation of gene expression
Describe packaging of DNA?
For prokaryotes
and eukaryotes
- associated with nucleoside proteins in the cytoplasm, tightly coiled by topoisomerase enzymes
- Within the nucleus, DNA is winded around a core complex of 4 types histones in a octamer structure forming a nucleosome
These nucleosomes are associated to a form a second level of packaging termed a chromatin fibre, which in of themselves can further be folded and looped forming chromosome structures
What enzyme is used to unwound DNA
and what proteins ensure that the single strands are prevented from reannealing?
DNA helices
SSBs
What group on primer is vital for the DNA polymerase III to add nucleotides?
3’-OH
RNA primers are removed by?inprocaryotes
DNA pol I
In regard to DNA protection for bacteria what two compounds are included in a restriction modification system of type 2 to proofread replicated DNA sequences?
DNA methylase
restriction endonuclease
that recognises short (4–6 bp)
palindromic sequences and cleaves foreign unmethylated DNA at a particular target
sequence
Messenger RNA is synthesised by RNA polymerase __,
while RNA polymerases I and III
catalyse the synthesis of :
II
rRNA (I), tRNA and snRNA (III).
In eukaryotes the upstream promotor element responsible for initial
recognition and binding of RNA polymerase to the TATA box in DNA is called?
CAAT box
Transcription of a eukaryotic gene results in the production of a
hnRNA, undergoes three events:
capping
polyA-tail exon splicing
Transcription of a eukaryotic gene results in the production of a ___ before mature mRNA is formed.
hnRNA, undergoes three events:
capping 5’-P group on G residue
polyA-tail on 3’-OH by poly (A) polymerase
Intron splicing mediated by snRNA’s found in spliceesome
In translation of mRNA what three stages are there?
Initionation
elongation
termination
One control mechanism that aid in the modulation of mRNA, responsible of degrading mRNA, what is it called?
RNA interference.
What type of enzyme have been widely exploited in molecular biology to enable the construction of recombinant
DNA, throu cleavage in a defined manner?
restriction endonucleases creating staggered ends or flush-ended.
In the extraction process of DNA when we’re talking about recovering DNA from cells, what compound is usually present when we cell rupture?
EDTA, which chelates
the Mg 2+ ions needed for enzymes that degrade DNA (termed DNases)
how do we degrade cell walls if present, when the cell is ruptured?
and what should we do with the cell membrane?
digested enzymatically (e.g. lysozyme treatment of bacteria)
solubilise with detergent
When we try to isolate DNA, the first step is to be sure to prevent the DNA from fragmenting by mechanical shearing, thus we want to choose the gentles possible method of rupture, so at what temp should this be performed?
4
After release of nucleic acids from the cells, RNA can be removed by treatment with?
and how about protein?
Finally, the DNA preparation is mixed with two volumes
of absolute ___, and the DNA allowed to precipitate out of solution in a
freezer.
- RNase
- by shaking the solution gently with water-saturated phenol, or with a phenol/
chloroform mixture, either of which will denature proteins, but not nucleic acids. - ethanol
How do we check for integrity of DNA?
agarose gel electrophoresis
Regarding UV spectroscopy, what ratio of absorbances between 260 and 280 nm tell us that the sample(DNA) is free of protein contamination?
1.8
What’s the differentiating factor one should keep in mind when we talk about differences between RNA and DNA isolation?
That RNA is very vulnerable to digestion by RNases, thus gloves should be worn, and a strong detergent should be included in the isolation medium to immediately denature any RNases.
In the case of isolating eukaryotic mRNA from a mixture of total RNA molecules, which technique can we use, if we note that we can make use of the fact of the presence of a poly(A) tail binds to the complementary oligo(dT) molecules of the column at high salt conc.
affinity chromatography
what type of gel electrophoresis can we use to separate large fragments of DNA, like chromosomes?
pulsed-field
following electrophoresis of DNA in horizontal agarose gels, give an example of an staining dye?
and how does it dye?
SYBR safe
it interchelates by insertion between the stacked base-pairs
After electrophoresis of DNA, how are these desired fragments removed (3)?
- physically by an scalpel
- glass-rod-crushing in a small volume buffer using agarose to digest the agarose
- electroelution
Gel electrophoresis remains the established method for the separation and analysis
of nucleic acids. However, a number of automated systems using pre-cast gels
and standardised reagents are available that are now very popular. This is especially
useful in situations where?
a large # of samples or high-throughput analysis is required
In regard to automated analysis of nucleic acid fragments, what 3 proposed methods are there?
- instruments employing micro fluidic circuits
- HPLC (which has gained popularity in DNA mutation analysis)
- MALDI-TOF
Explain Restriction mapping of DNA fragments?
how can this technique be used?
We analyse sizes that are produced by restriction enzymes in conjunction with bioinformatic analysis of the restriction fragment lengths to construct a map
by using restriction endonucleases we are able to distinguish which plasmids contain different bacterial strain ORFs.
identification of specific mRNA sequences of
a defined length by hybridisation to a labelled gene probe, is known as?
Northern blotting
How are a gene probe made?
by artificial chemical synthesis, by linking monomers of phosphoramadites forming an oligonucleotide
What is the most common compound that one may label DNA gene probes with?
how is this newly labelled probe, then purified?
32P, used in 5’ end-labelling (direct) carried out by alkaline phosphates followed by polynucleotide kinase
SEC
Explain Random Primer labelling of DNA?
DNA is first denatured, then placed in renaturing conditions with a mixture of hexamers that bind to DNA when finding complementary sequences, these hexanucleotides acts as primers for the synthesis of fresh strand of DNA catalysed by DNA polymerase since it has an exposed 3’-OH group
A further traditional method of labelling DNA is by the process of __ ___
Low concentrations of DNase I are used to make occasional single-strand nicks in the
double-stranded DNA that is intended as the gene probe
nick translation .
what type of probe is used to detect DNA amplification accomplished by the PCR
Molecular beacon probes
In PCR what stages are there in one cycle?
- Denaturation
- Annealing
- Extension
What factors do we have to consider wrt. the specificity of the PCR design regarding the design of the 2 primers?
These have to not only be complementary to sequences flanking the target DNA, but also
must not be self-complementary or bind to each other to form dimers since either
scenario prevents DNA amplifi cation. They must also be matched in their GC content
and have similar annealing temperatures in order to allow for a an optimum annealing
temperature
The annealing step allows the ____ of the two oligonucleotide primers, which are present in excess, to bind to their complementary
sites that flank the target DNA
hybridisation
The annealed oligonucleotides act as primers for DNA synthesis, since they provide?
a free 3 -hydroxyl group for DNA polymerase
what is the name of the common thermostable DNA polymerase used in PCR?
And what is its two benefits
taq DNA polymerase
Allows very high fidelity in regard to proofreading activity and ability to tolerate GC-rich DNA sequences.
Why would we use reverse transcriptase-PCR method (3)?
- To amplify RNA, by the use of reverse transcriptase
- Differentiate latent viruses (detected by standard PCR)
- Active viruses that replicate and thus produce transcription products
What’s the main drawback of PCR?
give 3 solutions
The exquisite sensitivity, since very large degree of amplification makes the system vulnerable to contamination.
_________
A. UV irradiation to damage already amplified products so that they can’t be used as templates
B. Incorporate Uracil and treat products with UNG, which degrades any PCR amplicons with incorporated uracil,
rendering them useless as templates
C. use of hotstart, to avoid mispriming, where reaction mixture is physically separated from DNA template – only when reaction begins does mixing occur.
What two types of DNA ends does restriction endonucleases cleave?
Sticky and flush
pairing of fragments derived from the same starting DNA molecules, termed ?
reannealing
When we have cleaved DNA fragments from the same starting DNA molecule, how do we stabilise the reannealing ?
Since the reannealing is are transient pairing owing to the weakness of H-bonds between the few bases in the sticky ends, we may use an enzyme called DNA ligase in a process termed ligation to form a 3’ -> 5’ phosphodiester linkage.
Why is the ligation process carried out in 10 degrees Celsius?
in order to lower the kinetic energy of the molecules, otherwise we would have base-paired sticky ends parting before they have been stabilised by ligation
What two general types of gene libraries are there? Why would done use either of these two?
genomic:
to understand the control of protein production for a particular gene
cDNA:
production of new or modified proteins or the determination of tissue-specific expression and timing patterns
______
I have to study this more before exam!!
Genomic libraries are constructed by isolating the complete _____ DNA from
a cell, then digesting it into fragments of the desired average length with restriction
endonucleases
chromosomal
Describe the complex process by which one make cDNA?
- Reverse transcriptase / buffer / dNTPs + primer (3)
- poly(dC) tail is added to its 3’ end, using terminal transferase and dCTP. also adds poly(dC) onto poly(A) of mRNA
- Alkali hydrolysis –> making a single strand cDNA
Now we make other complementary strand.
- add oligo (dG) primer to poly (dC) tail catalysed by Klenow fragment
How do we treat Blunt cDNA ends before eventual ligation?
nucleic-acid linkers with one internal site for a restriction endonuclease
Name a enrichment method for RNA
sucrose density gradient centrifugation
When do we want to use subtractive hybridisation?
to isolate mRNA transcripts that translate genes that are transcribed in a specific cell type or differently during a particular stage of cellular growth, frequently at very low levels
What two cloning methods for PCR are there?
- blunt-ended:
Where one make use of the 3’ overhanging A residue making it possible to clone the PCR product into dT vectors, followed by DNA ligase to ligate. - cohesive-ended cloning:
oligonucleotide primers are designed with a restriction endonuclease site incorporated into them
One key method of efficiently cloning DNA that obviates the need for restriction enzymes is termed the ___ ____
Gibson assembly
What is the major difference between cloning and PCR?
By clon- ing, it is possible to not only store a copy of any particular fragment of DNA, but also produce virtually unlimited amounts of it.
The vectors used for cloning, how can these vary?
- In their complexity,
- Their ease of manipulation
- Their selection
- The amount of DNA sequence they can accommodate (the insert capacity)
What two factors relate the choice and trade-off between various vector types for gene library contractions (2)?
- ease of the manipulations
- maximum size of foreign DNA insert of the vector
Many bacteria contain an extrachromosomal element of DNA, that carries genes for antibiotic resistance, conjugation or the metabolism of ‘unusual’ substrates. What is this element of DNA called?
natural plasmid
An uptake of cloning vector by bacteria is a process termed?
transformation
What does a bacterial origin of DNA replication (ori) ensures?
that the plasmid will be replicated by the host cell.
Some replication origins display stringent regulation of replication, in which rounds of replication are initiated at the same frequency as cell division. However, in the case for most plasmids, they have a relaxed origin of replication, what is the outcome of this?
that the rounds of replication is not tightly linked to cell division, thus plasmid replication will be initiated far more frequently than chromosomal replication.
Linearising a plasmid allows for?
a fragment of DNA to be inserted and the circle closed.
Describe insertional inactivation?
At which an DNA fragment insert can be detected by loss of resistance to antibiotic since some sites are places within the antibiotic resistance gene.
Two genes coding for resistance to antibiotics have been introduced into a cloning vector, what does these two genes individually allow for?
the first one allow for selection of cells that have plasmids, the other one can be used for detection of those plasmids that contain inserted DNA.
Insertional inactivation is a useful selection method for identifying?
recombinant vectors with inserts.
After which one has made a ligated DNA, i.e. recombinant vector with insert, what prior treatment do we need to introduce in order to make the cells competent before transformation
Ca2+ at 4 *C
How does one transform the competent cells with the small and circular recombinant vectors with ligated inserts?
with heat shock
A. After a brief incubation to allow expression of the antibiotic resistance genes, the cells are plated onto medium containing the antibiotic.
When colonies are replica plated, what does this pertain to?
B. Does replica plating illustrate the importance of a second gene for antibiotic resistance in a vector?
A. We’re able to distinguish between those colonies containing plasmids with inserts and those that simply contain re-circularised plasmids.
Thus colonies that grow on ampicillin but not on tetracycline must contain plasmids with inserts.
Since, the Bam HI site lies within the tetracycline resistance gene, this gene will be inactivated by the presence of an insert.
B. Yes
If the digested plasmids are treated with the enzyme alkaline phosphatase prior to ligation, what happens?
recircularisation will be pre- vented, since this enzyme removes the 5′-phosphate groups that are essential for liga- tion
Although recircularised plasmid can be selected against by the use of replica plating approach, its presence decreases the yield of recombinant plasmids containing inserts. How can we remedy this?
By treating the digested plasmids with alkaline phosphatase prior to ligation, re-circularisation will be prevented
the most popular restriction sites are concentrated into a region termed the?
multiple cloning site (MCS)
This blue/white selection method and insertional inactivation of antibiotic resistance genes do not, however, provide any information on the character of the DNA insert, just the status of the ___
vector
Apart from plasmids, we have virus-based vectors, in which instance should we use these?
When we want to propagate larger fragments (Inserts greater than 5 kb) of DNA in bacterial cells.
What type of vector is derived from the commonly used derived from λ bacteriophage?
And what advantage does this offer in comparison to plasmid cloning vectors?
- virus-based cloning vectors.
- approx. 16-fold advantage in cloning efficiency
What two types of replication modes does the bacterial DNA follow, wrt. λ bacteriophage.
- Lysogenic life cycle
- Lytic life cycle
When phage are subsequently released from the cell by lysing the cell membrane to infect further E. coli cells nearby. At the extreme ends of phage λ are 12 bp sequences termed cos (cohesive) sites, what do these allow for?
allow the phage DNA to be circularised, in order to protect the viral genome ends from nucleases in host organism.
Describe the process of vitro packaging, wrt. phage λ virus-based cloning vectors?
For the cloning of long DNA fragments, up to approx. 25 kb, much of the non-essential λ DNA that codes for the lysogenic life cycle is removed and replaced by the foreign DNA insert. The recombinant phage is then assembled into pre-formed viral protein particles
vitro packaged recombinant phage λ ( a virus-based cloning vector) are used to?
To infect bacterial cells that have been plated out on agar.
The recombinant aspect, denoting the non-essential λ DNA that codes for the lysogenic life cycle is removed, making it possible; Once inside the host cells, the recombinant viral DNA, to be replicated solely on the merit of normal lytic growth AND not on the lysogenic mode path, where we rely on integration, replication and cell division for the cloned vector to multiply.
In general, what two types of λ phage vectors have been developed?
λ insertion vectors and λ replacement vectors
When is the process of subcloning sometimes necessary ?
when the manipulation of a gene fragment cloned in a general purpose vector needs to be inserted into a more specialised vector for the application of techniques such as in vitro mutagenesis or protein production.
If we want the combined properties of phage and plasmids, what type of cloning vector should we use?
phagemid
What type of cloning vectors is ideal for techniques such as chain termination sequencing and in vitro mutagenesis
phagemid
what are cosmic-based vectors particularly useful for?
the analysis of highly complex genomes
The advantage of vectors that accept larger fragments of DNA than phage λ or cos- mids is that?
fewer clones need to be screened when searching for the foreign DNA of interest
DNA fragments with repeat sequences are sometimes difficult to clone in bacteria-based vectors, but may be successfully cloned in ___ systems
YAC
What’s the main advantage of YAC-based vectors?
And what two projects is this type of vector the main choice for?
- ability to clone very large fragments of DNA, as in the case of foreign DNA fragments up to 2000 kb
- in genome mapping and sequencing projects.
Plasmids used for cloning DNA in eukaryotic cells require (2)?
a eukaryotic origin of replication and marker genes that will be expressed by eukaryotic cells.
At present, the two most important applications of plasmids to eukaryotic cells are for cloning in?
yeast and in plants.
Why have so many cloning vectors have been developed for E. coli ?
- because due to the ease of growth and short doubling time we’re able to perform Initial recombinant DNA experiments
- the reliable competency factor regarding gram-positive bacteria wrt. extraneous plasmid DNA
- The natural ability of bacteriophage to introduce DNA into E. coli
What is the process by which recombinant molecules can be delivered into animal cells?
transfection
How can we increase the efficiency of the process of transfection?
by first precipitating the DNA with Ca2+ or making the membrane permeable with divalent cations
What compound can be used to maximise the uptake of DNA, wrt. transfection and as an selection marker?
PEG
What alternative method can be used to introduce DNA into animal cells?
electroporation:
the cells are subjected to pulses of a high-voltage
gradient, causing many of them to take up DNA from
the surrounding solution
Describe lipofection?
The recombinant DNA is encapsulated by lipid-coated particles that fuse with the lipid membrane of cells and thus release the DNA into the cell
The ability to deliver recombinant molecules into plant cells is not without its problems. Generally, the outer cell wall of the plant must be stripped, usually by enzymatic digestion, to leave a ______. In these cases transformation may also be achieved by?
- protoplast
- electroporation
Based on which principle is a clone containing the desired fragment chosen to be isolated, from the library?
hybridisation
Explain colony hybridization?
Colony:
colony growth
replica plating
lysis
denature liberated DNA and bound to membrane
membrane incubation with pre-hybridisation mix to
block non-specific site
add gene probe
membrane wash
autoradiography
Why would we consider PCR screening over gene hybridisation of gene libraries?
- rapidity
- indication of the size rather than the sequence of the cloned insert
In which case would we consider immunological screening rather than gene hybridization in reagrd to cDNA libaries.
In this case what type of a vector would be utilise to prepare a specially adapted vector which transcribes and translates any cDNA inserted into it
- When the sequence is required is partially characterised for a certain protein, we may go for antibodies to screen a protein of interest.
- expression
what is protein engineering?
involves a logical sequence of analytical and computational techniques centred around a design cycle for producing site-directed mutations. This includes the biochemical preparation and analysis of proteins, the subsequent identification of the gene encod- ing the protein and its modification. The production of the modified protein and its further biochemical analysis completes the concept of rational redesign to improve or probe a protein’s structure and function
What two examples of methods are part of rational designed cycle system?
Kunkel Oligonucleotide-Directed Mutagenesis
PCR mutagenesis
error prone PCR is an accelerated evolutionary approach to protein engineering and has been useful in the production of ?
novel antibodies produced by phage display
catalytic antibodies
For a foreign gene to be expressed in a bacterial cell, why is it important to choice right promotor sequences upstream of the coding region?
for correct and efficient transcription, the sequence and position are specific to a particular host
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 ?
regulatory sequence.
Why is it usually essential to use cDNA instead of a eukaryotic genomic DNA to direct the production of a functional protein by bacteria.
because bacteria can’t remove introns
What does the signal sequence do?
it directs protein to a particular cellular compartment or even out of the cell altogether into the supernatant
What three methods can be used to indetify and analyse mRNA?
quantitive PCR
northern blot
RPA
What does in situ hybridisation make it possible to determine?
the chromosomal location of a particular gene fragment or gene mutation by the use of a radio labelled DNA or RNA probe
An alternative labelling strategy used in karyotyping and gene localisation is fluorescence in situ hybridisation (FISH), what’s the advantage of this method?
is that separate gene regions may be identified and com- parisons made within the same chromosome preparation, using different gene probes that are labelled with different fluorophores, each specific for a particular chromosome
what method can be used to detect:
1. viral gene expression
2. tissue-specific gene expression
- RT-PCR
- Differential display
The binding of a regulatory protein or transcription factor to a specific DNA/RNA site results in a complex that may be analysed by the technique termed?
EMSA
As a result of the production of phagemid vectors and as a means of overcoming the problems of screening large numbers of clones generated from genomic libraries of antibody genes, a method for linking the phenotype or expressed protein with the genotype has been devised. This is termed ___ ___
phage display
Most methods used in identifying regulatory sites in DNA rely on the fact that the interaction of a DNA-binding protein with a regulatory DNA sequence will protect that DNA sequence from degradation by an enzyme such as DNase I
What assay (incl. gel electrophoresis and autoradiography) can we use to elude the position of the protein-binding sequence within the DNA from the size of the fragments either side of the footprint region.
footprint
In addition to the detection of DNA sequences that contribute to the regulation of gene expression, an ingenious way of detecting the protein transcription factors has been developed. This is termed the?
yeast two-hybrid system
There are a number of ways of experimentally changing the expression of genes. Traditionally, methods have focussed on altering the levels of mRNA by manipulation of promoter sequences or levels of accessory proteins involved in control of expression.
In addition, post-mRNA production methods have also been employed, such as __ ___ underlying the process of interference RNA induced by siRNA’s
antisense RNA
Mutations occurring within exons may alter the amino acid composition of the encoded protein by causing amino acid substitution or by changing the reading frame used during translation
give an method that can be used to detect these point mutations directly without examine the PCRs without gel electrophoresis?
What if the point mutation is unknown, which method can we use?
allele-specific oligo- nucleotide PCR (ASO-PCR)
denaturing gradient gel electrophoresis
Polymorphisms are particularly interesting elements of the human genome and as such may be used as the basis for differentiating between individuals. All humans carry repeats of sequences known as minisatellite DNA, of which the number of repeats varies between unrelated individuals. Hybridisation of probes that anneal to these sequences using ___ ____ is one method to type and identify those individuals
give a PCR based alternative that can detect locu of hypervariable minisatellites throughout the genome?
Southern blotting
MLPA
A number of disease loci have been identified and located to certain chromosomes. This has been facilitated by the use of in situ mapping techniques such as FISH. In fact, a number of genes have been identified and the proteins of interest determined where little was initially known about the gene except for its location
Which PCR- based technique can be used to map the putative gene (for which a function has yet to be assigned) to a chromosomal region ?
expressed sequence tag (EST)
Molecular genetic analysis has allowed a discrete set of cellular genes, termed ______, to be defined that play key roles in events of elucidation of diseases that are multifactorial and involve a significant contribution from environmental factors
oncogenes
