Textbook Notes (363,534)
Canada (158,402)
Biochemistry (180)

Topic 26.docx

5 Pages
Unlock Document

Western University
Biochemistry 2280A
Christopher Brandl

Recombinant DNA Technology  Genetic Engineering Definition: The techniques by which DNAfragments from different sources (different chromosomes, different organisms, or man-made) are recombined to make a new DNAmolecule How to Clone Your Favourite Gene (YFG) ● Many aspects of modern biotechnology require high level expression of a protein product ● This is most conveniently done by expressing a recombinant form of the gene in E.coli Plasmids ● To introduce YFG into E.coli, you need a vector or vehicle to allow its introduction, selection, and propagation in the “host” organism; plasmids serve as these vehicles ● Aplasmid is a generally small circular DNAthat replicates independently of the bacterial chromosome ● Depending on the plasmid, the copy number per cell can vary but it can be in the range of 100 copies per cell ● The key features of a plasmid that make it useful for cloning are: ○ one or more useful restriction sites at which YFG can be inserted ○ a selectable marker to allow the identification of the plasmid in bacteria; the most common selectable markers for use in bacteria are genes encoding resistance of antibiotic ○ an origin of replication to allow replication of the plasmid, independently of the host chromosome Synthetic Oligonucleotides ● Several applications of recombinant DNAtechnology that we will use in cloning YFG require the use of synthetically made single stranded DNAs; these include: ○ hybridization probes, primer for DNAsequencing, PCR, and mutagenesis PCR (Polymerase Chain Reaction) ● PCR is a technique to produce large amounts of a specific DNAfragment from any DNA source in which it is found as little as one time; it is the exponential amplification of DNAby repeated extension of 2 primers ● The steps are as follows: ○ Denature the DNAtemplate at 90-90 C o o ○ Anneal the two primers at 50-60 C; you must have sequence information to make the primers o ○ Extend the primer with a DNApolymerase and the 4 dNTPs at 72 C ○ Repeat the first three steps ~30 times Gel Electrophoresis ● Different DNAfragments are most easily separated by size; sizing of DNAis most easily accomplished by gel electrophoresis using polyacrylamide or more often agarose as the separating matrix ○ the agarose acts as a molecular sieve in which the DNAmolecules snake through the pores ● Smaller DNAmolecules move more easily through the pores and thus can migrate faster ● The DNAis pulled through the gel matrix by placing the gel in an electric field and the DNAmigrates towards the positively charged area as it is itself negatively charged ○ DNAhas a uniform mass to charge ratio thus it is solely the differential sieving based on the size of the DNAthat is responsible for separation ● To visualize the DNAon the gel, we can use ethidium bromide (EtBr) which will fluoresce red in UV light ○ the degree of staining is proportional to the amount of bound EtBr which is proportional to the mass of each DNAfragment ○ if amount of DNAis low, EtBr staining does not have sufficient sensitivity ● When th32EtBr does not work (DNAtoo low), the DNAis then radioactively labeled using P ● Labeling the ends of the DNAallows their detection after exposure of the gel to X-ray film cDNA: ● cDNAis a complementary DNAcopy of a RNA; it is synthesized using Rna as a template and the enzyme reverse transcriptase (it requires a primer) ● When using mRNA as the template poly-dT is often used as the primer for the synthesis of the cDNA as it will anneal to the polyA tail ● cDNAs are very useful for expressing proteins since unlike genomic DNA, the cDNA lacks introns; thus when used for PCR, the second strand does not have to be synthesized ● If one wishes to make dsDNAfrom initial single stranded cDNA, DNApolymerase is used Hybridization ● Hybridization is a tool to detect a DNAsequence; the Tm is the temperature at which 1/2 of the specific double stranded DNAhas become single stranded What determines Tm? ● Intrinsic factors ○ A:T/ G:C ratio: thei more G:C, the higher the TM ○ Length of the helix, shorter molecules melt at lower temperatures ● Extrinsic factors ○ Counter-ions neutralize the negative charge in the phosphate backbone - reduce repulsion; higher salt raises the Tm ○ Solvent - formamide and urea will decrease Tm by weakening H-bonds ○ Note also that imperfect matches have lower Tm than perfect matches Melting is Reversible ● The renaturation requires appropriate conditions, salt, and slow cooling ● Any 2 nucleic acid strands will hybridize if they share sufficient complementarity; ssDNAwill anneal with ssDNAand RNA; RNA will anneal with RNA Types of Blots ● Southern Blots uses a DNAprobe and DNAfilter; Northern blots use a DNAprobe and RNAfilter ● Steps in a Northern blot for determining if a gene important in muscle development is expressed in other cell types are as follows: ○ Isolate RNAfrom different tissues (muscle, liver, spleen etc.) ○ Separate RNAon basis of size by agarose gel electrophoresis ○ Transfer the separated RNAto a membrane ○ Prepare a radiolabeled probe for the fragment of interest; if the probe is dsDNA, it must be denatured; if ssDNAor RNA, it must be the complementary ○ Incubate the HOT probe with the filter ○ Wash away the nonspecifically bound probe ○ Expose the probed filter to x-ray film ○ Bands will appear in the lanes of tissues in which the gene is expressed Restriction Enzymes ● Identification, characterization (sequencing), and manipulation (mutagenesis, overexpression) of a gene requires that a gene be resolved from other genes ● The restriction enzymes most commonly used are Type II enzymes which show specific recognition and specific cleavage of DNA ● These restriction enzymes are site specific DNAbinding proteins which in general recognize and cut palindromic sequences most commonly o
More Less

Related notes for Biochemistry 2280A

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.