Manipulating genes and cells Flashcards
What is recombinant DNA/ gene splicing?
The ability to isolate a given piece of DNA out of millions of nucleotides and to be able to generate new DNA molecules in the test tube and to introduce the custom made material back into the living organisms
How can a gene be isolated and purified?
Restriction nucleases
A nuclease catalyzes the hydrolysis of a phosphodiester bond in a nucleic acid. But these enzymes have a property distinct from other nucleases, they cut double-stranded DNA only at particular sites. Thus restriction nucleases can be used to produce a reproducible set of specific DNA fragments from any genome.
Restriction nucleases cleave DNA at specific nucleotide sites.
These nucleases are usually obtained from bacteria and their names reflect their origins: for example, the enzyme Eco RI comes from Escherichia coli.
How are fragments of DNA separated
Using gel electrophoresis, which separates fragments on the basis of their length.
The mixture of DNA fragments is loaded at one end of a slab of agarose or polyacrylamide gel, which contains a microscopic network of pores. A voltage is then applied across the gel slab. Because DNA is negatively charged the larger fragments migrate more slowly because their progress is impeded more by the agarose matrix.
How is DNA then sequenced after separation?
The most widely used scheme for doing this is the dideoxy method which is based on DNA synthesis carried in vitro in the presence of chain terminating dideoxyribonucleosidetriphosphates. In this technique, DNA polymerase is used to make partial copies of the DNA fragment to be sequenced. These DNA replication reactions are performed under conditions that ensure that the new DNA strands terminate when a given nucleotide (A, G, C or T) is reached. This method produces a collection of different DNA copies that terminate at every position in the original DNA, and thus differ in length by a single nucleotide.
What’s a powerful fact that allows us to detect specific nucleotide sequences in both DNA and RNA
The fundamental capacity of a single-stranded nucleic acid molecule to form a double helix only with a molecule complementary to it
Describe what nucleic acid hybridization is, in the context of sickle cell anemia.
DNA is extracted from fetal cells, treated with restriction nucleases and then electrophoresed through the gel. Two DNA probes are used to test the fetal DNA, one corresponding to the normal b-globin gene sequence in the region of the mutation, and the other corresponding to the mutant gene sequence. If the probes are short (about 20 nucleotides) they can be hybridized with DNA. Using this technique it is possible to distinguish whether DNA isolated from the fetus contains, one, two, or no defective b-globin genes. For example, a fetus carrying two copies of the mutant b-globin gene (one from each of two chromosomes) (which will result in the disease)can be recognized because its DNA will hybridize only with the probe that is exactly complementary to the mutant sequence
What does cell cloning mean?
The act of making many identical copies of a DNA molecule
Give a method for DNA cloning and briefly describe it
Transformation
Introduce the DNA to be copied into a rapidly dividing bacterium.
Once inside the recipient cell, the donor DNA can become a part of the recipient genome(through the process of homologous recombination) or in special cases – can be maintained as a piece of DNA independent of the bacterial chromosome
Why might recombinant DNA be put into an independent molecule, rather than with bacteria chromosomes, when cloning?
May find it easier to manipulate, copy and purify recombinant DNA
For this, we use plasmids - small circular DNA molecules that can replicate inside a bacterial cell
Why is a plasmid perfect for being a cloning vector?
A plasmid vector contains a replication origin (a particular sequence in a genome at which replication is initiated which enables it to do so.
It also has a cutting site for a convenient restriction nuclease, so that the plasmid can be opened and a foreign DNA fragment can be inserted.
Plasmids also usually contain a gene for some selectable properties such as antibiotic resistance which enables bacteria that take up the recombinant DNA to be easily identified.
Figures showing cloning using a plasmid vector
and then:this DNA fragment can then be recovered by cutting it cleanly out of the plasmid DNA usingthe appropriate restriction enzyme and separating it from the plasmid DNA bygel electrophoresis
What’s a genomic library?
Human genomic libraries can be constructed using restriction nucleases and ligase. A genomic library comprises a set of bacteria, each carrying a different small fragment of human DNA.
How do you find a specific gene in a genome library?
A DNA probe for that gene
Describe how to create a DNA probe
Here we work in reverse, using the amino acid sequence from isolated VIII protein. Based on this sequence (partial one) a nucleotide sequence was prepared chemically to synthesize the DNA probe. A complimentary VIII fragment was the unidentified using hybridization
We now know that the Factor VIII gene (180000 nucleotide pairs) also contains many introns (regions that are not translated into proteins), how can we create library which does not have introns?
This is called a cDNA library (complementary DNA library).
To create a cDNA library the total mRNA is extracted from the cells at hand and a DNA copy of this is made using reverse transcriptase. Following this DNA polymerase is used to make a complementary DNA strand. Since the m-RNA only contains protein information and is thus devoid amongst others of intron information, the DNA produced will have no introns in its sequence, this is cDNA. now we can clone cDNA as we described for genomic DNA before. The factor VIII sequenced void of introns was then identified by using a portion of the genomic factor VIII DNA as a probe.
Figure of a bacterial colony carrying a particular DNA clone can be identified by hybridization.
Describe how PCR works
PCR is based on the use of DNA polymerase. The polymerase is guided to the sequence to be copied by short primer oligonucleotides that are hybridized to the DNA template at the beginning and end of the desired DNA sequence. Because the primers need to be chemically synthesized, PCR can be used only to clone DNA whose beginning and end sequences are known. Guided by these primers DNA polymerase is then used to make many copies of the sequence required in vitro. Note that special heat-resistant DNA polymerase from a thermophilic bacterium is used so that it will not denature as the DNA strands are heated after each cycle to create single strands that are then used again by the polymerase for further synthesis.
What can PCR be used for?
Detecting the presence of a viral genome in a sample of blood.
Because of its ability to enormously amplify the signal from every single molecule of nucleic acid, PCR is an extraordinarily sensitive method for detecting trace amounts of virus in a sample of blood or tissue without the need to purify the virus.
Show the process of splicing together fragments of DNA from different sources.
After each DNA insertion step, the recombinant plasmid is cloned to purify and amplify the new DNA. The recombinant molecule is then cut once with a restriction nuclease, as indicated, and used as a cloning vector for the next DNA fragment.
How can large amounts of protein be produced from DNA cloning?
Protein-coding DNA sequence cloned into an expression vector and introduced into cells.
A plasmid vector has been engineered to contain a highly active promoter (refresh: a promoter is a region of DNA that facilitates the transcription of a particular gene), which causes unusually large amounts of mRNA to be produced from an adjacent protein-coding gene inserted into the plasmid vector. Depending on the characteristics of the cloning vector, the plasmid is introduced into bacterial, yeast, insect or mammalian cells, where the inserted gene is efficiently transcribed and translated into protein.
