Biotechnology Flashcards
What is a recombinant plasmid?
It’s a plasmid with foreign DNA being inserted into it.
What do restriction enzymes do?
These cut DNA molecules at specific DNA sequences called restriction sites. This results in restriction fragments.
What part exactly do restriction enzymes cut?
They cut sugar-phosphate backbones.
What is a cloning vector?
It is the original plasmid.
What is a genomic library?
A genomic library is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome. ( a genomic library that is made using bacteriophages is stored as a collection of phage clones)
What is a bacterial artificial chromosome (BAC)?
It is a large plasmid that has been trimmed down and can carry a large DNA insert. BACs are another type of vector used in DNA library construction.
What is the cDNA library (complimentary DNA library)?
It is a library that contains genes that are expressed in the cell at a given point of time.
It is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell.
What are the differences between genomic DNA library and cDNA library?
- Genomic DNA library is a representation of all genes, cDNA library is a representation of genes that are expressed. (So cDNA is much smaller)
- Genomic DNA library contains the sequences for both intron and exon. cDNA library contains only exons. (cDNA has less sequence information compared to a genomic DNA library)
- No. of recombinants to be screened is less in cDNA.
How to extract gene of interest?
With a nucleic acid probe having a sequence complementary to the gene. This process is called nucleic acid hybridisation.
How scientists overcome the problem of difficulties that hinder expression of cloned eukaryotic genes in bacterial host cells?
They employ an expression vector, which is a cloning vector that contains a highly active bacterial promoter.
How biologists avoid eukaryote-bacterial incompatibility?
They use eukaryotic cells such as yeasts al hosts. But even yeasts may not possess the proteins required to modify expressed mammalian proteins. So cultured mammalian or insect cells may be used to express and study proteins.
From which electrode DNA segments move in gel electrophoresis?
From negative cathode to positive anode.
What variations in DNA sequence are called?
Polymorphisms
What sequence changes that alter restriction sites are called?
Restriction fragment length polymorphisms
How can we determine the function of the gene?
Disable it and observe the consequences. Introduce mutations into a cloned gene using in vitro mutagenesis, and so, alter or destroy its function. When returning the mutated gene to the cell, normal gene’s function might be determined by examining the mutant’s phenotype.
Gene expression can also be silenced using RNA interference (RNAi). Synthetic double stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA.
How can we find which gene is causing a “disease”?
Genetic markers called single nucleotide polymorphisms occur on average every 100-300 base pairs. These can be detected by PCR and any SNPs shared by people affected with a disorder but not among unaffected people might show the location of the disease-causing gene
What is Southern blotting?
It’s a technique used to identify DNA fragments by combining gel electrophoresis and nucleic acid hybridisation.
How can we test changes in the expression of a gene during embryonic development?
-northern blotting
-reverse transcriptase-polymerase chain reaction
Both methods are used to compare mRNA from different development stages
Identification of mRNA at a particular development stage suggests protein function at that stage
How can genetic profiles be analysed? Mention 2 ways
Using RFLP analysis (restriction fragments length polymorphisms) by southern blotting
Even more sensitive is the use of genetic markers called short tandem repeats which are variations in the number of repeats of specific DNA sequences (PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths)
How can we measure the expression of thousands genes at one time?
UsingDNA microarray assays. This compares patterns of gene expression in different tissues, at different times, or under different conditions.
How dies cloning occur?
The nucleus of an unfertilised egg cell is replaced by the nucleus of a differentiated cell
Do clones look exactly like their “parent”?
No, not necessary. They are genetically identical but behaviour and appearance might differ due to different expression of genes.
What is the most commonly used vector for introducing new genes into plant cells?
Ti plasmid
What is the difference between tori potent and pluripotent cells?
Totipotent cell can differentiate into a complete organism. Pluripotent cell can differentiate into all or almost all cell types in an organism but not into an entire organism.
Describe the chain termination method (Sanger method)
- Denaturati in of DNA via heat
- Now we have a template strand
- Add template strand with primer to DNA polymerase and free nucleotides (dNTPs).
- Then add modified nucleotides (ddNTPs).
- Sample of each reaction is taken and tested via gel electrophoresis.
