Lecture 4 - Selection, Screening, DNA Sequencing Flashcards
What is the difference between selection and screening?
• Selection refers to picking out only the transformed cells containing vectors
• Screening refers to vector identifying transformants carrying the recombinant vector containing gene of interest
How is selection carried out?
• Achieved by selecting for the trait expressed by the vector under peer selection pressure
• Cloning vectors comprised of the antibiotic resistance gene (ie ampicillin resistance)
• Bacteria cells transformed with plasmid vector (containing ampicillin resistance) are plated on the agar media containing Ampicillin
• Only the cells with the plasmid vector survive due to the expression of ampicillin antibiotic resistance genes
How is selection used for fungi?
• For fungi, make use of auxotrophic marker genes, which encode critical enzymes for amino acid synthesis
• The corresponding host is an auxotrophic mutant for the amino acid tryptophan
• Host cell incapable of growing on the minimal media
• Growth can be achieved by enriching the media with the tryptophan
• Host cells transformed with the vector containing the Trpl gene, can grow in the minimal media
How is screening of recombinant clones carried out?
• Gene of interest and plasmid vector are ligated
• Possibility of obtaining three different products during the process
• Self-ligated vector (vector without gene)
• Vector with the gene of interest (recombinants)
• Gene—gene ligated product (if single RE is used)
Screening of recombinant clones
What is negative selection?
• Selection is made for the loss of gene product encoded by the vector
• Vector genes are inactivated by inserting foreign DNA segments into the vector
• Two approaches
1. Indirect (replica plating)
2. Direct (chromogenic) approach
Adapted from: https://blog.addgene.org/pIasmids-101-positive-and-negative-selection-for-
plasmid-cloning
How is antibiotic selection by replica plating performed?
• pBR322 plasmid vector encodes for two antibiotic resistance genes - ampicillin and tetracycline
• Cells transformed with pBR322 vectors can grow on media containing both the antibiotics
• Gene of interest is inserted within any one of these antibiotic resistance genes
• Recombinant clones remains tolerant to one antibiotic and sensitive to the other antibiotic
• The recombinant clones do not survive on the media containing tetracycline, they cannot be selected directly
• Replica plating technique
• Transformed cells are plated on a media containing ampicillin (master plate) first
• Impression of the colonies is traced by keeping the nitrocellulose membrane
• Place impression on ampicillin and tetracycline containing replica plate
Only non-recombinants can grow (recombinants
identified by comparing replica plate with master
plate
Advantage
• Simple
Limitation
• Transformants grow slower in amp and tet containing plates
than amp plate alone.
• Replica plating is cumbersome
What is the chromogenic method?
• Distinguish recombinants from non-recombinants based on color of the colony
• Blue-white screening technique
• Makes use of the ß-galactosidase enzyme encoded by E. coli and converts lactose into glucose and galactose
• Active ß-galactosidase enzyme can hydrolyze X-Gal (an analog for lactose) into insoluble deep blue-colored products
• Plasmid vectors synthesize a short segment of the first 146 amino acids of the ß- galactosidase enzyme encoded by the lacZ gene
• The E. coli host cells used to carry such vectors are mutant for lacZ gene (lacZAM15 deletion mutation)
• Independently the vector and the host cannot synthesize a functional enzyme,
• When the host carries such vectors, due to a- complementation, the functional ß- galactosidase enzymes are synthesized
• A multiple cloning site (MCS) is present within the lacZ sequence in the plasmid vector
• This sequence can be nicked by restriction enzymes to insert the foreign DNA
• When a plasmid vector containing foreign DNA is taken up by the host E. coli, the a- complementation does not occur
• Functional ß-galactosidase enzyme is not produced
What are the limitations of blue-white screening?
• The blue-white technique is only a screening procedure; it is not a selection technique
• The lacZ gene in the vector may sometimes be non-functional and may not produce ß- galactosidase. The resulting colony will not be recombinant but will appear white
• Even if a small sequence of foreign DNA may be inserted into MCS and change the reading frame of lacZ gene. This results in false positive white colonies
How to screen for specific gene of interest?
• Identify the clones with a target gene/specific DNA segment among the large pool of recombinant clones carrying unique DNA segment/gene in them
• Cell-specific cDNA library
• Represents only the genes that are expressed in the particular cell type can be constructed which is cellular specific
• Cell-specific cDNA library can still be significantly large
• Require sensitive, precise, and high throughput screening strategy to identify the target clones
• These methods include:
1. Nucleic Acid Hybridization
2. Colony or Plaque Hybridization
3. PCR-Based
Screening for specific gene of interest
What is nucleic acid hybridization?
• Design probes that bind with its complementary target sequence
• DNA/RNA probes are single-stranded molecules of around 50 to 500 bp length and are labeled with either radioisotopic or non-radioisotopic molecules
• Enzymes attached to probes for catalyzing reactions that produces color
or fluorescence for detection
• Identification of any target sequence from any library
Screening for specific gene of interest
What is PCR-based screening?
• Colonies from the master plate are maintained as pooled colonies
• Cells are lysed, and this pooled mixture of DNA is used as a template to carry out PCR using a set of target DNA-specific primers
• PCR using the same set of primers for individual colonies from the master plate, which are part of the positive pool
What are the advantages and limitations of PCR-based screening?
Advantages
• Colony PCR is the quickest method for screening recombinant clones
• The primers specific to the gene of interest can be used to confirm the
gene of interest
Limitations
• It is based on specific primer designing, the primers need to be designed with high precision and accuracy.
• Can be challenging when you do not have the exact gene sequence
How is analysis of recombinant clones carried out?
• After identifying the recombinant clone, it is necessary to go for a detailed characterization of the gene
• Hybridization techniques help ascertain the presence of part of the DNA sequence of interest
• To know the exact coding sequences, presence of regulatory elements, and any other features, sequencing is required
• Sequencing is the most accurate way to cross-check the recombinant clone and verify the sequence of interest
What is DNA sequencing?
• Sequencing DNA means determining the order of the four chemical building blocks
• Establishing the sequence of DNA is key to understanding the function of genes and other parts of the genome
Sanger sequencing
Massively parallel DNA sequencing
Nanopore DNA sequencing
What is Sanger sequencing?
• Sanger sequencing involves making many copies of a target DNA region
• DNA polymerase enzyme
• Primers
• Four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
• Template DNA to be sequenced
• Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP),
• Each labeled with a different color of dye in much smaller amounts
• Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl available
• No further nucleotides can be added
• Chain ends with the dideoxy nucleotide is marked with a particular color of dye
• Mixture is first heated to denature the template DNA
• Mixture is cooled so that the primer can bind to the single-stranded template
• Temperature is raised again allowing DNA polymerase to synthesize new DNA
• DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide
• Strand synthesis end with the dideoxy nucleotide
• Repeated in many cycles
• By the end of many cycles tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original
• Ends of the fragments will be labeled with dyes that indicate their final nucleotide DNA
