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Suggest why plasmids cannot be used to insert genes directly into plant cells.
- -Plasmids cannot enter plants cells through the cellulose cell walls
- -Plasmids are not integrated into the host cell genome and will thus be degraded.
- -Plasmids belong to the prokaryotic system which would not be recognised or expressed by an eukaryotic cell.
State and explain 3 essential features of a plasmid for it to be used as a vector for gene cloning.
- 1) Possess an origin of replication-allows for the replication of itself along with the inserted DNA
- 2) Has at least one known restriction site - allows for the insertion of DNA fragments
- 3) Have genetic markers- allows for the selection and identification of cells which have taken up the recombinant DNA
Explain the presence of restriction enzymes in bacteria.
- -Restriction enzymes cuts and digest any foreign viral DNA that invades the bacteria cell
- -This protects the bacteria from any viral attack
Explain why the restriction enzymes can digest viral DNA but not the bacteria's genome.
- -The bacterial genome is protected from degradation by its own restriction due to
- -the addition of a methyl group (-CH3) to an adenine or cytosine at the restriction site
- -the viral DNA however is generally not methylated and thus would be degraded by the restriction enzymes
Briefly describe the role of DNA ligase and reverse transcription in the formation of recombinant DNA.
-DNA ligase catalyst the formation of phophodiester bond between the nucleotides of the foreign DNA and the plasmid.
-Reverse transcriptase catalyses the formation of cDNA from the single-stranded mRNA transcript.
Explain why restriction enzymes that result in the formation of sticky ends are more preferably compared to those that results in blunt ends.
Restriction enzymes that results in the formation of blunt ends require an additional step using terminal transferase to add nucleotides to add sticky ends in the gene of interest to allow for the annealment of the DNA fragment and vector plasmid.
Explain why a plasmid might be a more advantageous vector compared to a bacteriophage in making a DNA library.
- -Allows for easy selection of bacteria with recombinant DNA as there is presence if genetic markers in plasmids compared to bacteriphage DNA
- -It is easier to replicate plasmid vector compared to viral DNA as there is an origin of replication in plasmids unlike that in viral DNA
Define the term gene probe and state its function
- A gene probe consists of a short sequence of radioactively-labelled nucleotides which is complementary to part of the DNA sequence of interest and thus binds with the sequence.
- The radioactivity acts as a marker which can then be detected by autoradiography.
Outline how a recombinant DNA molecule ,using a plasmid as the vector, is formed.
- -The DNA containing the gene of interest and the plasmid are cut with the same restriction enzyme to generate restriction fragments with complementary sticky ends.
- -The restriction fragments and cleaved plasmids are then mixed
- -The complementary sticky ends will then anneal via complementary base-pairing.
- -DNA ligase is then added to catalyse the formation of phosphodiester bond between the gene of interest and the plasmid to form the recombinant DNA
State the advantages of PCR.
- -Allows a particular gene to be amplified in large amounts
- -A faster method compared to cloning using recombinant bacteria. It can amplify DNA exponentially within hours
- -A specific process that targets only the desired DNA to be copied
- -Only minute amounts of DNA need to be present in the starting material
Explain the two factors that result in the separation of the DNA molecules in nucleic acid gel electrophoresis.
1) Size of the DNA molecules-the smaller molecules are able to move through all the agarose gel faster than the larger ones and hence would travel further down the agarose gel compared to the larger molecules
2) Length of the DNA fragment-longer fragment will migrate less along the gel compared to shorter DNA strands
State the function of the following in nucleic acid gel electrophoresis.
ii) DNA ladder
i) Glycerol is a dense liquid, thus it helps to prevent DNA from being washed away by the buffer solution and ensures that the DNA sinks into the well
ii) The ladder is composed of known standards, so when the sample undergoes electrophoresis, the size or weight of each sample can be estimated
Describe how a genomic DNA library is produced.
- -A genomic DNA library is a complete set of recombinant plasmid clones, each carrying copies of a particular DNA segment from the initial genome,
- forming a genomic library
- -DNA from an organism or a particular cell type is extracted and digested with restriction enzymes to form DNA fragments .
- -Each cut fragment is then spliced into a plasmid vector to form a recombinant plasmid and is used to transform a bacteria host.
- -Each transformed bacteria cell is then cultured where it multiply into a colony containing a fragment of the DNA from the genome of the organism of interest.
Explain why reverse transcription of mRNA is more often used to isolate foreign DNA to be inserted into prokaryotic genome.
- -Reverse transcription of mRNA can be used to obtain eukaryotic genes that excludes introns from mRNA
- -The presence of introns in eukaryotic genes generates problems for expressing these genes in a prokaryotic system
- -Bacteria cells lack a RNA-splicing machinery and thus introns in eukaryotic genes cannot be spliced out and will render the final protein non-functional
- -By obtaining cDNA from mRNA, the introns are absent and thus results in the formation of a functional protein when expressed using a prokaryotic system
Describe the conditions necessary for transformation.
- -The bacterial cells must first be treated with CaCl2 solution so as to increase their competence (ability to take up extracellular DNA)
- -The bacteria cells must then be subjected to a brief heat shock to make the pores of the cell membrane to appear transiently so that the cell membrane will be permeable to the plasmids.