Chapter 12. Cloning Techniques PDF

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Lecture notes over chapter 12: cloning techniques...

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Chapter 12 Biochemistry Thursday, October 8, 2020

10:23 AM

What Does it Mean To "Clone"? - Clone: a collection of molecules or cells, all identical to an original molecule or cell - "cloning a gene" --> make many copies of it - Cloned gene can be exact copy of natural gene or an altered version of natural gene - Recombinant technology makes cloning possible Plasmids Are Very Useful in Cloning Genes - Plasmids--> naturally occurring, circular, extrachromosomal DNA molecules, these extra genes can help code for certain beneficial metabolic processes, like ability to break down a certain antibiotic ○ Can be cleaved by restriction enzymes, and artificial plasmids can be made by inserting new DNA fragments and ligating it together. ○ Can also be autonomously replicated and propagated in bacteria - Cloning Vectors--> are plasmids that can be modified to carry new genes - Useful plasmids as cloning vectors have: ○ Replicator- this is an origin of replication(ori) ○ Selectable marker- antibiotic resistant gene usually, this is usually how plasmid cells are selected for, everything else dies in the antibiotic except this plasmid ○ Cloning Site- site where restriction enzymes can cleave in the DNA plasmid that won't disrupt replication or inactivate essential markers. Virtually Any DNA Sequence Can Be Cloned(BIG POWER OF DNA RECOMBINATION) - Nuclease cleavage at a restriction sites "opens" circular plasmid so foreign DNA can be inserted here - Ends of linearized plasmid joined to the ends of the fragment so that circle can be closed again, creating a recombinant plasmid - Recombinant plasmids--> hybrid DNA molecules consisting of plasmid DNA sequences + inserted DNA elements(called inserts). Also referred to as chimeric constructs, or chimeric plasmids - Recombinant plasmid can replicate when placed in a suitable bacterial host. - Difficult to clone inverted repeats, origins of replication, centromeres, telomeres - Only limitation is the size of foreign DNA segment, plasmids w/ inserts larger than 10kbp aren’t replicated efficentl - Short synthetic DNA duplexes whose nucleotide sequence contains a little more than a restriction site can be blun end ligated onto any DNA. Linkers--> short DNA described above

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Directional Cloning - To insert foreign DNA in a particular orientation ○ Cleave the plasmid w/ two different restriction enzymes

○ Cleave the foreign DNA w/ the same two restriction enzymes ○ Foreign DNA can only be inserted in one direction now Biologically Functional Chimeric Plasmids - Plasmids can be used to transform recipient E.Coli cells - Transformation--> uptake an replication of exogenous DNA by a recipient cell - Selection of clones can be based on bacteria growth in certain antibiotic medias - Useful upper limit on cloned insert in plasmids --> 10,000 bases pairs… bacteria will delete nonessential gene whe put under selection pressure(inserted genes are non-essential). Most eukaryotic exceed this size.

What is a DNA Library? - Genomic Library--> set of cloned fragments that collectively represents the genes of a specific organism, particu genes can be isolated from the library(like getting books from a library) you just have to know where to look - Any gene is only a very small part of an organism's genome so it is impractical to recover such rare sequences directly from isolated nuclear DNA, instead a genomic library is made - Preparing a genomic library ○ Isolating total DNA from organism ○ Digesting DNA into fragments of suitable sizes ○ Cloning the fragments into an appropriate vector - Creating a DNA library--> DNA library contains as many genes from the organism of interest as possible ○ Step 1) Isolate the genomic DNA from the organism of interest ○ Step 2) Digest the DNAs w/ a restriction enzyme (4-6 nucleotide specific)  The DNA can be cut into fragments much smaller than the average gene size. Digestion is carried out for a brief period that leaves many of the restriction sites uncut, called a partial digest ○ Step 3) Choose a suitable vector for the required insert size and cut w/ a restriction enzyme that produces compatible stick ends ○ Step 4) Ligate the digested genomic DNA and the vector ○ Step 5) Transform the library into bacterial host cells  A large number of transformed bacterial colonies must be isolated and kept to ensure that all possible gene from the genome of interest are represented on at least one vector - PCR is Used To Clone and Amplify Specific Genes ○ PCR(Polymerase chain reaction)--> technique for identifying and dramatically amplifying the amount of a specific DNA segment. This is the preferred cloning strategy ○ PCR relies on DNA polymerase to carry out multiple rounds of DNA synthesis ○ Design of appropriate primers is a key to PCR--> must be complimentary to the strand in which it is desired to amplify. ○ PCR protocols are automated by carrying out the reaction in a thermal cycler.

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- Identifying Specific DNA sequences by Southern Blotting (Southern Hybridization) ○ Southern Blot--> technique that uses gel electrophoresis combined w/ labeled probes for a DNA sequence of interest that allows for the detection of repeat expansions within specific genes. ○ Step 1) Gel Electrophoresis--> DNA fragments("the library") are fractionated by size w/ agarose gel electrophoresis ○ Step 2) Blotting  Separated molecules are blotted to an absorbent support and then incubated w/ labeled(radioactive or something) oligonucleotides. ○ Step 3) Hybridization  Detection of the label shows the location of DNA fragments that hybridized w/ the probe

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○ Northern blotting--> use of the Southern technique to identify particular RNAs following the separation by gel electrophoresis, blotting, and probe hybridization ○ Western Blotting---> probe of choice, antibody, identifies a specific protein Complimentary DNA(cDNA) Libraries are DNA Libraries Prepared from mRNA(In Cell Cloning) - cDNA are DNAs copied from mRNA templates - cDNA libraries are made by synthesizing cDNA from purified cellular mRNA - Most eukaryotic mRNA carry 3'-poly(A) Tails--> allows for mRNA to be selectively isolated from preparations of tota cellular RNA by oligo(dT)-cellulose chromatography

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Reverse Transcriptase-PCR - Reverse Transcriptase--> enzyme that makes a DNA strand by copying RNA as the template - DNA polymerase then copies this DNA single strand and makes another strand so it could end up being a double strand. - In RT-PCR--> RNA population is converted to cDNA by RT, and then the cDNA is amplified by PCR. The cDNA amplification step provides the original RNA sequence information. The CDNA also serves as a template for furthe cycles of PCR - Applications of RT: Detection of expressed genes, examinations of transcript variants, generation of cDNA templates for cloning and sequencing Measuring The Abundance of an mRNA by RT-qPCR - Real- time quantitative polymerase chain reaction(RT-qPCR) is a technique used for determining mRNA expressio w/o cloning its gene. - This technique measures the abundance of a particular mRNA within the total RNA from a particular cell type - RT is used to make cDNA copies of the mRNA within the sample, and PCR is used to amplify the amount of this cDNA - Measuring the amount of DNA formed provides information about the amount of target mRNA in the original RNA sample

DNA Microarrays Are Arrays of Different Oligonucleotides Immobilized On A Chip - Robotic methods can be used to make combinatorial libraries of DNA oligonucleotides directly on a solid support - The completed library is a 2-D array of different oligonucleotides - Final product of such procedures are referred to as "gene chips" because the sequences synthesized upon the chip represent the sequences of chosen genes - The oligonucleotides on such gene chips are used as probes in hybridization experiments to reveal gene expressio patterns. Can The Cloned Genes in Libraries Be Expressed? - Expression vectors--> engineered so that any cloned insert can be transcribed into RNA and in some instances to proteins.

Reporter Gene Constructs - Reporter Gene Constructs--> chimeric DNA molecules composed of gene regulatory sequences positioned next t an easily expressible gene product. - Reporter gene--> a gene that is placed upstream of potential regulatory regions of genes so this region can be investigated. Expression of reporter gene serves as a report on the effectiveness of the regulatory element - GFP(green fluorescent protein)--> a gene with many advantages - Detection of GFP only requires irradiation w/ near-UV or Blue light.

Identifying Protein-Protein Interactions 10...