Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
27 Cards in this Set
- Front
- Back
|
What is recombinant DNA? |
joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. |
|
|
What are recombinant proteins? |
an outcome of the expression of recombinant DNA inside living cells. |
|
|
What assists with making the recombinant protein? |
Various kits are commercially present to assist both the cloning of the gene, and the following recombinant protein fabrication. |
|
|
What is the difference between the two types of producing recombinant protein? |
molecular cloning incorporates the replication of the DNA within a living cell, whereas polymerase chain reaction (PCR) replicates DNA in the test tube, without living cells. |
|
|
What is DNA replication? |
the process of making an identical copy of a section of double-stranded DNA |
|
|
How is cloning done? |
- a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes - cut at specific 4-8bp - and then united with a plasmid that has been cut with the same restriction enzymes. - When the fragment of chromosomal DNA is joined with its cloning vector, it is called a "recombinant DNA molecule." - Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA. |
|
|
What is a plasmid? |
self-replicating extra-chromosomal circular DNA molecules, |
|
|
What are the steps for cloning? |
Preference of host organism and cloning vector, - Preparation of vector DNA, - Preparation of DNA to be cloned, - Formation of recombinant DNA, - Foreword of recombinant DNA into the host organism, - Selection of organisms having recombinant DNA, - Screening for clones with preferred DNA inserts and biological characteristics. |
|
|
When is shotgun sequencing used? |
When sequencing long DNA strands |
|
|
Describe shotgun sequencing? |
- DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads. - Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing. Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence |
|
|
Describe Sanger sequencing? |
chain termination method, is a technique for DNA sequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication |
|
|
What is the limitation of Sanger sequencing? |
can only be used for fairly short strands 100-1000 bp |
|
|
What is the role of the vector? |
The vector carries the piece of foreign DNA to the host cell |
|
|
What is an expression system? |
A protein production system that is achieved by manipulating gene expression in an organism such that it expresses large amounts of a recombinant gene. |
|
|
Which proteins and enzymes are used to make expression systems? |
- DNA polymerase - reverse transcriptase for RNA analysis - restriction endonucleases for cloning |
|
|
What is the most important post-translational modification for proteins? |
Glycosylation |
|
|
Give some examples of where glycan is required for the biological function of proteins? |
- Fc effector function og IgG - regulated clearance of the hormone lutropin (LH) |
|
|
How does glycosylation affect lutropin? |
When lutropin is glycosylated with native alpha and beta subunits it is fully active. When on of its subunits is deglycosylated its receptor binding activity is significantly reduced. |
|
|
What 5 questions must be thought about when choosing an expression system? |
1) What type of protein do i want to make? 2) Is the protein soluble? 3) Does the protein need post-translational modification? 4) What is the codon usage of my protein? 5) Is the product toxic to the host? |
|
|
What complications may arise from question 1? |
If you can choose an expression system that already makes your protein of choice, there will be far fewer complications with q 2,3,4,5 - If there is not a compatible expression system then in most cases E.coli would be used as it is the simplest and most understood. |
|
|
What complications may arise from question 2? |
Insoluble aggregates frequently form if the protein does not fold properly. A fusion protein could be used to over come this problem. Glutathione-S-transferase (GST) or maltose binding protein (MBP) |
|
|
What complications may arise from question 3? |
- Post-translational modification can be very species specific - Removal of the N-terminal methionine (all organisms can do this), N-glycosylation, or O-glycosylation (only done by eukaryotes). |
|
|
What complications may arise from question 4? |
- Different species have different codings for each amino acid, eg. CTT is the triplet codon for leucine in humans, but threonine in yeast. So if you took the human genome and put it into yeast the amino acid sequence would have different shape - The bioinformatics must be sufficiently done to select the best species with the correct amount of tRNA for making the desired protein, if not the product will not be compatible. |
|
|
What is the rate limiting factor for the protein expression in q4? |
The percentage frequency of each amino acid in the chosen expression system species. |
|
|
What happens if the amino acid frequency is limited? |
The error rate and interrupted translation gives truncated protein products due to - frame shifting - misinterpretation leading to complete inhibition and no protein product. |
|
|
What complications may arise from question 5? |
The protein product may be toxic to the host if the host would not normally make it. In this case the host may treat the product as a prion (infectious protein particle). The protein may start to aggregate together and poison the rest of the cell. The host then limits transcription and translation and self destructs. |
|
|
What are the factors that determine the type of expression system used? |
- Time spent expressing the proteins - Ease of handling the expression system - Amount of protein needed/yield - Destination of expressed protein |
