Recombinant DNA technology

Recombinant DNA
1. molecules that are artificially made in labs and are not natural
2. technology that is used to create and study these hybrid molecules constructed from DNA obtained from different biological sources
Cloning in host cells
1. 1.DNA to be cloned is isolated and treated with a restriction enzyme to create fragments ending in a specific sequence 2.The fragments are then linked to plasmid molecules that have been cut with the same restriction enzyme creating a collection of recombinant vectors 3.The recombinant vectors are transferred into E. Coli host cells which inside replicates to form many copies or clones. 4.The bacteria are plated on nutrient medium, where they form colonies, and are screened to identify those that have taken up recombinant plasmids
2. Yeast is widely used as a host for cloning and expressing eukaryotic genes
Cloning without host cells
1. Polymerase Chain Reaction
  1. PCR copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities
  2. PCR requires two oligonucleotide primers (anneal to denatured DNA) one complementary to the 3' end of one strand of the DNA to be amplified and one complementary to the 3' end of the other strand (complementary strands are synthesized by a heat-stable DNA polymerase)
  3. three steps are denaturation, primer annealing, and extension, which are repeated to exponentially amplify the DNA
Recombinant libraries
1. Genomic library
  1. contains at least one copy of all the sequences in the genome of interest
  2. number of clones needed to give certain probability of containing all genomic sequences is calculated as N=ln(1-P)/ln(1-f), where N is the number of required clones, P is the probability of recovering a sequence, and f is the fraction of the genome in each clone
2. cDNA library
  1. contains complementary DNA copies made from the mRNAs present in a cell population
  2. RT-PCR can be used to generate cDNA from mRNA by first making a single-stranded cDNA copy of the mRNAs using reverse transcriptase, and then using PCR to copy the single-stranded DNA into double-stranded DNA
3. Specific clones can be recovered from a library
  1. probe is any DNA or RNA sequence that has been labelled in some way and is complementary to some part of a cloned sequence present in the library
  2. to screen a plasmid library, clones from the library are grown on agar plates to produce colonies and then a filter is hybridized with a nucleic acid probe to the DNA sequence of interest, and the corresponding colony to the one identified on the filter by the probe is identified and recovered
Analysis of cloned sequences
1. Restriction map
  1. establishes the number, order, and distance between restriction sites on a cloned DNA segment
2. Southern blot
  1. identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest
3. Northern blot
  1. involve RNA bound to a filter and provide information regarding the expression of specific genes and the transcript sizes
4. DNA sequencing
  1. most common method of DNA sequencing is dideoxy chain termination sequencing developed by Sanger
  2. large-scale genome sequencing is automated and uses fluorescent dye labelled dideoxynucleotides
  3. genomes of numerous prokaryotes and eukaryotes, including humans, have been fully sequenced
Basic procedure for recombinant DNA technology
1. 1.DNA to be cloned is purified from cells or tissues 2.Restriction enzymes are used to generate specific DNA fragments 3.The fragments produced are joined to other DNA molecules that serve as vectors creating a recombinant DNA molecule 4.The recombinant DNA molecule is transferred into a host cell where it replicates/clones of the rDNA 5.As host cells replicate, the cloned DNA molecules within them are passed on to all their clones creating a population of host cells 6.The cloned DNA can be recovered from host cells for use in commercial applications or in research
Recombinant DNA molecules are constructed from several components
1. Restriction enzymes
  1. produced by bacteria and they restrict or prevent viral infection by degrading DNA of invading viruses
  2. they cut the genome into smaller fragments that can be manipulated, separated, copied and analysed individually
  3. it binds to DNA and recognises a specific nucleotide sequence (recognition sequence) and cuts both strands of the DNA within that sequence
  4. they are useful because, in cloning derives, of their ability to accurately and reproducible cut genomic DNA into fragments (restriction fragments)
2. Vectors
  1. they are carrier DNA molecules that can replicate cloned DNA fragments in a host cell
  2. they must be able to independently replicate and should have several restriction enzyme sites to allow insertion of a DNA fragment
  3. a plasmid is an extra chromosomal double-stranded DNA molecule that replicates independently in bacterial cells
    1. plasmids used for DNA cloning have been engineered to contain a number of convenient restriction sites and a marker gene to select for its presence in the host cell

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Recombinant DNA technology

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	Criado por Abbey Roberts aproximadamente 11 anos atrás