Isolating,Cloning and Sequencing DNA

*Outline the procedures for cloning an eukaryotic gene in a bacterial plasmid. (p10-16)

(I)ris (I)s (T)oo (S)elf (C)entered (I)n (P)erformance Isolation Insertion Selection Cloning Identification Production 1. Isolation of plasmid DNA and human DNA Plasmid vector: -Plasmid is obtained from E coli which carries two useful marker genes amp^R and lac Z -amp^R allows bacteria to be resistant to the antibiotic ampicillin -lac Z codes for the enzyme ß-galactosidase which catalyses the hydrolysis of lactose -this plasmid has only one restriction site and it lies within the lac Z gene. It recognised by the restriction enzyme used in the next step. Human DNA: -Insulin mRNA is extracted from pancreatic cells (ß-cell of Islets of Langerhans) -Reverse transcriptase added to extract uses the mRNA as a template to form cDNA -DNA sequences/Linkers containing the appropriate restriction site are ligated to the ends of cDNA -the Linkers are cut by restriction enzyme in the next step to produce sticky ends 2. Insertion of human DNA into plasmids (D)igest (M)ix (F)orm -Bacterial plasmid and cDNA of insulin gene are digested the same restriction enzyme- to produce complemetary sticky ends Restriction enzyme cuts the plasmid DNA at its single restriction site, disrupting lac Z gene -cDNA of insulin gene and the digested plasmids are mixed to allow sticky ends of DNA from both sources to anneal by complementary base pairing -Mixture incubated with DNA ligase Annealed strands are joined covalently by phosphodiester bonds, forming recombinant plasmid. Ligation mixture will contain 3 major types of DNA molecules: reannealed plasmids, reannealed cDNA fragments and Recombinant plasmid 3. Transformation - Cells are electroporated hence making pores appear transiently in the cell membranes of the bacterial cells, making the membrane permeable to DNA and allowing DNA molecules to enter the cells. (refer to fig 9 on p11) 4. Selection and Cloning of transformed cells with recombinant plasmids -Transformed cells are placed onto nutrient agar plates containing the ampicillin and X-gal and incubated at 37°C -Only bacteria transformed with the plasmid (reannealed and recombinant) will grow to form a colony as they have the amp^R gene that confers amplicilin resistance -During formation of the recombinant plasmid, insertion of cDNA into the plasmid within the lac Z disrupted the lac Z gene hence enzyme ß-galactosidase not produced and X-gal not hydrolysed thus bacterial colony carrying recombinant plasmid will appear White hence are picked since they contain recombinant plasmid with cDNA for insulin gene -Bacterial colonies carrying reannealed plasmid will synthesised ß galactosidase which will hydrolyse the X-gal to produce blue pigment -As bacteria multiply, plasmids within bacteria will multiply as well 5. Identification of bacterial colonies carrying gene of interest with Gene Probes -White colonies containing recombinant plasmids are identified and grown on an agar plate and then transferred onto a filter -DNA is then denatured with chemicals to separate the two strands -radioactive DNA probe which is ssDNA complementary to a portion of the gene of interest is added to the filter. It will anneal to gene of interest by H-bonds -Black spots formed on film during AUTORADIOGRAPHY show the positions of where the probe has bound to the gene of interest -Based on the location of black spots, we can go to the original master plate to pick the colonies that are confirmed to have the gene of interest inserted into the plasmid. (ALTERNATIVE METHOD OF SELECTING RECOMBINANTS: using 2 anitbiotic-resistant genes(amplicilin and Tet^R where restriction site lies within Tet^R) (must refer to pg 14!) 6. Mass production of protein of interest -Bacterial colonies identified to carry the insulin gene are picked and grown in large quantities in a suitable medium which are then induced to produce large amounts of insulin. -The hormones produced can be extracted, purified and packaged for use by diabetics.

*Explain how eukaryotic genes are cloned using E. Coli cells to produce eukaryotic proteins to avoid the problems associated with introns. (p9)

-Instead of using eukaryotic gene, use the mRNA of the gene as introns have already been removed from the mRNA through RNA splicing -However mRNA are less stable than DNA as a genetic material, use Reverse transcriptase to convert RNA into DNA then insert that DNA into vector

Describe how reverse transcription avoid the problem of eukaryotic introns. (p9)

-Reverse transcription is the process in which RNA is used as a template to make a complementary strand of DNA -Procedures: 1. Isolate mRNA of desired gene from appropriate cell 2. Add Reverse transcriptase which will use the mRNA as a template to make a complementary DNA strand 3. Add RNAseH to degrade mRNA strand 4. Add DNA polymerase which will use cDNA to synthesize the second DNA strand 5. Double stranded cDNA produced--->insert into plasmid vector

Define Genetic Engineering.

-is the process of changing the genetic make-up of a living organism or cell by artificial means, such as the introduction of a gene from another species or the introduction of a mutation into a specific gene -is used for: gene cloning mass production of useful proteins producing gene library for gene therapy

List some examples of vectors.

-plasmids of bacteria (e.g E coli) -viral DNA of lambda bacteriophage -yeast artificial chromosome

Extra

DNA ligase- seals nicks present in a DNA molecule by catalysing the formation of phosphodiester bonds between adjacent nucleotides in a DNA molecule. Vector- a DNA molecule which carries our target DNA into host cell and allows the foreign DNA to be reproduced(cloned) in large quantities. Cloning vector- is a vector that carries our target DNA into the host cell and allows the foreign DNA to be reproduced in large quantities Vector are taken into host cell where the recombinant DNA can be replicated or recombinant protein can be produced.

What are the problems of cloning a Eukaryotic gene using bacteria? (p9)

F(reaking) IMPT Foreign Introns mRNA Promoter Translatable 1. Eukaryotic proteins may be recognised as foreign material by bacterial proteases and degraded Solution: Use a strain of bacteria that lack these proteases as host cell 2. Eukaryotic genes contain introns while bacterial gene do not hence bacteria unable to excise introns from RNA and the eukaryotic introns will be used in translation causing wrong a.a to be incorporated into the protein 3. Bacteria do not have cellular Machinery to modify the protein after translation Solution: Use a eukaryotic host cell 4. Eukaryotic promoters not usually recognised by bacterial RNA polymerases Solution: Eukaryotic gene must be linked to a bacterial promoter so that bacterial RNA polymerase can bind and transcribe the inserted gene 5. mRNA transcribed from eukaryotic gene may not be translatable on bacterial ribosomes Solution: Use a eukarytoic host cell e.g yeast

*Describe the properties of plasmids that allow them to be used as DNA cloning vectors.

ROM (Remain, Origin of rep, Marker gene) 1. Able to enter and remain in the host cell: -as vector is derived from host itself hence is recognised by host and will not be destroyed thus able to carry the desired gene into the cell and allow the gene to be replicated and passed to daughter cells 2. Contain one Origin of replication -thus enables vector together with DNA fragment inserted into it to be replicated by host cell's DNA polymerase. 3. Contain Marker gene that will give a visible phenotype to the bacteria this is easily detected -thus allows for identification and selection of bacterial host cells which have taken up the vector molecule which carries our desired gene

*Describe the natural function of restriction enzymes (aka Restriction Endonucleases).

• The role of restriction enzymes in bacteria: Protects the bacteria from foreign DNA (e.g viral DNA) as the enzymes destroy foreign DNA. (Also digests DNA molecules into restriction fragments. Are very specific, recognising and binding to restriction sites in DNA molecules. Binds to the restriction site and break the covalent phosphodiester bonds at specific points in both strands to give either sticky ends or blunt ends ) • Structure of restriction site: Palindromic • Difference between blunt ends and sticky ends: Sticky ends are staggered cut with single-stranded ends while blunt ends are simple cut across both strands at a single point. *complementary base pairing by H bonds first followed by formation of phosphodiester bond

*Explain the formation of recombinant DNA molecule.

• The use of the same restriction enzyme for both vector and DNA molecules: Restriction enzyme binds to the restriction site in DNA breaks phosphodiester bonds at specific points in both strands gives rise to either sticky ends or blunt ends DNA fragment added from another molecule cut by same enzyme • The process of annealing between complementary sticky ends by formation of H bonds: sticky ends anneal together by forming H bonds to complementary sticky ends from the DNA fragment cut by same restriction enzyme. • The need for DNA ligase to form the phosphodiester bonds to join the plasmid and gene of interest: Through base pairing DNA ligase seals nicks present in the DNA molecule by catalysing the formation of phosphodiester bonds between adj nucleotides in a DNA molecule recombinant DNA molecule formed.