Recombinant DNA Introduction to Recombinant DNA technology
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1 Recombinant DNA Introduction to Recombinant DNA technology Restriction EnzymesMaking RecombinantsMolecular Techniques
2 I. Introduction to Recombinant Technology (ch. 19) What is a recombinant?Recombinant DNA refers to a combination of DNA molecules that are not found together in nature, produced by joining DNA obtained from different biological sourcesVector = carrier DNA molecules that transfer and replicate inserted DNA fragmentsPermit entry into host cell, where it can be replicated or cloned into many copiesHow do you use a vector to produce recombinants?Plasmids – simplest of vectors, contain an origin of replication (so it make copies autonomously in the cell)They also contain selectable markers!Can use restriction enzymes to insert segments of DNA from one source into a plasmid, it can then be taken up into the cell and cloned
3 Recombinant DNA technology Cutting DNA and pasting it in new combinationsDNA purifiedEnzymes generate fragmentsFragments inserted into vectorVector transferred to host cellAs host cell replicates, recombinant molecules are passed on to progenyCloned DNA can be recovered & analyzed – can be transcribed & translated in the host cell
4 II. Fragments generated using Restriction Enzymes Cleavage of recognition sitesHost cell protects its DNA via methylation
5 RE’s recognize specific Palindromic sequences = recognition sequences EcoRISmaIPstIoverhangs provide “sticky ends” that reanneal with complementary ss tails on other DNA fragments
6 Properties of REsNamed after the bacteria from which they were isolatedEcoRI = from E. coli, it’s in group IThree groups based upon the types of sequences they recognize, the nature of the cleavage made in the DNAcut genomic DNA into fragments called restriction fragmentsBlunt & Staggered CutsSmaI = blunt cutEcoRI = staggered cut (leaves a 5’ overhang)PstI = staggered cut (leaves a 3’ overhang)DNA ligation = in staggered cuts, if two compatible ends anneal, then DNA ligase seals the phosphodiester bond between the two DNA molecules
7 III. Making Recombinants: 1). Create Donor DNA fragment, via restriction digest2). Add fragment to a vector, sticky ends hybridize to vector and are pasted using ligase3). Donor DNA is replicated, transcribed and translated, and thus the recombinants can be selected.
10 Types of VectorsChoice of vector primarily depends on desired size of genomic DNA insertPlasmids (5-10 kb)pUC182686 bp, in host it replicates 500 copies per cellContains several RE sites & has the lacZ gene (selectable marker) allows recombinant plasmids to be easily identified.Bacteriophage (10-40 kb)Lambda (able to replace 1/3 of the chromosome with large pieces of foreign DNA)higher transformation efficiency: 1000X more efficient than plasmidsM13 - can easily be subjected to mutagenesisCosmids (50 kb)Hybrid vectors, part plasmid – part lambdaBACs & YACs (300 kb, up to 1,000 kb)Artificial chromosomes
12 The phage DNA is cut w/ a RE, producing chromosomal fragments – this is mixed with DNA from another source (also cut w/the same RE. Ligation produces recombinant vectors that are packaged into phage protein heads in vitro and introduced into bacterial host cells.Inside the host cells they replicate and form many copies of infective phage, each of which carries the insert.
15 Constructing a genomic library Cloning Vectors (i.e. Plasmids & Bacteriophages)Must have:1) origin of replication2) cleavage site3) gene whose product allows transformed cells to be distinguished (selectable marker)DNA libraryPool of all recombinant plasmids generated by ligating DNA fragments from a source of interest into a vectorcDNA libraryIsolated mRNA is reverse transcribed into dsDNA. This is then cloned into a vector to make smaller a cDNA library
16 Finding specific DNA molecules from a library Selecting for vectorsYou can plate the bacteria with a particular gene on a selective plate (use the selectable marker)e.g. if the colonies are white, they contain the insert… if blue – they don’t
17 B) Sequence probe: Colony Hybridization Screening a plasmid library to recover a particular sequence-Labeled probe (specific sequence)Hybridizes with DNA from colonies.Autoradiography used to detect hybridization.
18 Analyzing Cloned Sequences: Once a cloned sequence has been identified and selected from a library it can be used for many things:probe to find/study regulatory regionsinvestigate organization of the genestudy expression in cells/tissues
19 IV. Molecular Techniques Restriction Digests (RFLPs)PCRDNA Sequencing
20 1) Restriction Fragment Length Polymorphisms (RFLPs) Variations in DNA fragment length generated by cutting with a restriction enzymeRestriction mapping = Inherited as alleles & can be mapped to specific regions on individual chromosomes (used as markers)200 bp bp bp bp
21 Restriction Mapping look at the number order and distance between enzyme cutting sites along a cloned segment of DNA :
22 (2) Polymerase Chain Reaction - PCR Fast method for many copies of a specific segment of DNA…replication in vitro… Taq polymerase… specific primers…
23 Forensics applications: examining DNA markers to identify criminals
24 (3) DNA sequencingprocess for sequencing particular regions of DNA, 4 sequencing reactions made for each base
25 Chain termination method 1) primer annealed to a single strand of DNA (3’end) this is distributed into 4 tubes. Each tube has one of the 4 dNTPs modified as dideoxynucleotide (ddNTP) w/ label.2) DNA pol is added to each tube & the primer is elongated forming a complementary strand to the template.3) As synthesis takes place, the pol can insert a ddNTP instead of a dNTP, causing synthesis to STOP. As the reaction proceeds, the tube w/ ddATP will accumulate molecules that terminate at all positions containing A.4) The DNA fragments from each reaction tube are separated by gel electrophoresis & read from bottom to top!
26 Automated SequencingGels directly read into a computer- as the terminated DNA strands pass near the bottom of the gel, an argon laser excites the fluorescent dye attached to the ddNTP and it fluoresces at a specific wavelength.