Recombinant DNA technology Flashcards
Summary
Recombinant DNA technology
Applications in gene analysis
Applications in genome analysis
..
Recombinant DNA (rDNA) 5 points
- Is a DNA constructed in the laboratory from 2 (or more) natural or artificial DNA fragments.
- Digestion with the appropriate
restriction enzymes. - The purified fragments are ligated using a DNA ligase.
- Different DNA fragments can be successively ligated to produce rDNA
- rDNA is then cloned via insertion into a vector and amplification into
a host cell
Molecular cloning
5 points
- DNA fragment to be cloned + cut vector (with antibiotic resistance gene)
Ligation -> - Complex: vector/insert (DNA construct)
- Transformation: intro of DNA complex to host cells
- Selection of the transformed cells on a solid growth media containing the
selection agent. - Bacterial colonies containing the plasmid vector and the insert.
(Each colony is a clone containing
a single insert DNA.)
rDNA is used in basic science
- gene analysis
2. Genome analysis
rDNA is used in Biotechnology
3 points
- Production of recombinant proteins
- Production of Genetically Modified Organisms (GMO)
- Molecular vaccines
Gene isolation Complementary DNA (cDNA) libraries
Collections of bacterial clones containing cDNA representing expressed genes in a given tissue at specific conditions
and corresponding to a specific physiological situation.
Only expressed genes are represented.
Gene isolation
Genomic-DNA (gDNA) libraries
Collections of bacterial clones containing fragments of genomic DNA digested by a restriction enzyme.
The entire genome is represented: Coding sequences, Regulatory sequences, “Junk” DNA
Making cDNA libraries:
- RNA extraction and mRNA purification.
- cDNA synthesis and production of double stranded DNA.
- Ligation of the adapters and their digestion.
- Ligation of cDNA into the vector.
- Packaging of the rDNA (cDNA in the vector) into the phage capsule.
- Plating the phage library into host bacteria.
Making gDNA libraries:
- gDNA extraction and purification
- Digestion of gDNA and vector with a restriction enzyme.
- Ligation of DNA fragments into the digested vector.
- Packaging of the rDNA (gDNA fragments inserted in
the vector) into the phage capsule. - Plating the phage library into host bacteria.
Probes
3 points
- Labelled RNA or DNA fragments with a known sequence used to detect
a complementary sequence in a DNA or RNA population. - Homologous probes: from the same organism
Heterologous probes: from a different organism, used to detect
similar sequences in the studied organism.
- The isolated clone is then sequenced and the sequence
analysed.
Transcriptome (total expressed genes in a tissue) analysis
- Expressed Sequence Tags (ESTs): Random sequencing of thousands of clones from a cDNA library prepared from a given tissue at specific conditions.
Genome Analysis (entire genome.)
- Sequencing DNA libraries containing fragments representing the entire genome of a given organism.
- The obtained sequences are made available to the Scientific community via accessible databases: NCBI: National Center for Biotechnological Information
Mapping genomes
Assigning/locating a specific gene to a particular region of a
chromosome and determining the location and relative distances between genes on the chromosome.
Physical map
The physical, DNA-base-pair distances from one gene to another.
Genetic linkage map
Order of genes on a chromosome and the relative distances between these genes.
Associating gene with function/phenotype
Monitoring change in gene expression
2 points
- Single gene approach
Northern blotting.
Quantitative Reverse transcription-Polymerase Chain Reaction (QRT-PCR).
- Global approach
Checking the expression of thousands of genes at the same time.
Microarray technology
RNA sequencing
Associating gene with function/phenotype
Gene silencing - Knock down technologies - Antisense RNA
3 points
- Used to reduce the expression of a gene to check the impact on the physiology.
- Antisense RNA are RNA sequences complementary to the target RNA, they hybridize to it and block its translation.
- Antisense RNA are produced by transforming the target organism with
a cDNA that is transcribed into antisense RNA.
Associating gene with function/phenotype
Gene silencing - Knock down technologies - RNA interference using small RNA
3 points
- Small RNA are natural or synthetic double stranded RNA molecules of 20-25 nucleotides complementary to the target RNA.
- they hybridize to the target RNA and block its translation. They lead to its degradation by specific RNases.
- Antisense RNA and small RNA allow to identify gene function.
Associating gene with function/phenotype
Knock-out Technology
5 points
- Knock-out are collections of insertionnal mutants produced by rDNA technology.
- A vector is used to transfer randomly or specifically a tDNA
(transfer DNA) to a location in the genome to alter a gene at that locus. - Each mutant has an alteration in a single gene.
- By tracing the tDNA it is possible to find which gene was affected.
- By identifying the change in the phenotype it is possible to link gene with the function.
