Genetics and recombinant dna
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Genetics and Recombinant DNA. BIT 120. Cotton Pests. Cotton Bollworm. Cotton Pests. Cotton Leaf Perforator. How Do Farmers Deal With Pest Insects?. Chemical Control Biological Control. Recombinant DNA.

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Cotton pests
Cotton Pests

  • Cotton Bollworm


Cotton pests1
Cotton Pests

  • Cotton Leaf Perforator


How do farmers deal with pest insects
How Do Farmers Deal With Pest Insects?

  • Chemical Control

  • Biological Control


Recombinant dna
Recombinant DNA

  • Definition : DNA molecule produced artificially and containing sequences from unrelated organisms.

    • Genetic Engineering

    • Use of techniques involving recombinant DNA technology to produce molecules and/or organisms with new properties.

    • Biotechnology

    • All inclusive term for several technologies including but not limited to recombinant DNA. Refers to the use of technology in applications for solving fundamental problems in biology.


Restriction endonucleases
Restriction endonucleases

  • Also called restriction enzymes: digest DNA at specific sequences


Sequence recognition r e
Sequence Recognition -R.E.

  • · Restriction endonucleases -- cut double stranded DNA at specific sequences, protection against viruses in bacteria.

  • · Sequences often palindromes: a sequence which is the same when read in either direction. ”A man a plan a canal: Panama”




Dna ligase
DNA ligase

  • · DNA ligase joins 5'-phosphate and 3'-hydroxyl ends of DNA

  • · Two fragments formed by EcoRI can be rejoined by ligase.

  • Similarly, Eco RI fragments from two different pieces of DNA can be joined



  • Plasmids
    Plasmids

    • · Extrachromosomal, circular small (2-3 kb) DNA in a bacterial cell which can replicate independently but which cannot integrate into the host chromosome.

    • · Drug resistance plasmids are not essential for the cell's growth, but confer antibiotic resistance.

    • · Plasmids used for molecular cloning have been artificially created by recombining fragments of various existing plasmids.

    • · Plasmids contain multiple cloning sites with several restriction endonuclease sites.





    Creating a recombinant dna molecule
    Creating a Recombinant DNA molecule

    • · A plasmid (vector) is digested with EcoRI at a single site to produce two sticky ends.

    • · A sample of human DNA is also digested with EcoRI to produce pieces with the same sticky ends

    • · Human DNA- or cDNA copied from mRNA using reverse transcriptase from retroviruses.

    • · The two samples are mixed and allowed to hybridize, some molecules will form with pieces of human DNA inserted into the plasmid vector at the EcoRI site.

    • · DNA ligase is used to covalently link the fragments.



    Inserting recombinant dna into host
    Inserting recombinant DNA into Host

    • Transformation

    • cell made competent to take up DNA

    • competent cells: electroporation – poke holes in membrane and calcium chloride- make cells more permeable to DNA

    • Transfection

    • when the cloning vector used has aspects of a virus, the host cell can be infected (transfected) to insert the recombinant molecule

    • Electroporation

    • the cell is placed in an electric field such that small pores are temporarily opened in the membrane. Added DNA can enter through these pores.



    Selection
    Selection

    • Antibotic resistance

    • · Plasmid vector contains an ampicillin resistance gene making the cell resistant.

    • · Growth of transformed cells (cells receiving the plasmid) can be identified on agar medium containing (e.g.) ampicillin.



    Further selection
    Further selection

    • · The plasmid vector contains another identifiable gene (e.g., a second drug resistance or an enzyme activity), with the coding sequence of this gene containing the restriction site for insertion.

    • · Insertion of the foreign DNA at this site interrupts the reading frame of the gene and result in insertional mutagenesis.

    • · In the following example, the -galactosidase gene is inactivated. The substrate "X-gal" turns blue if the gene is intact, ie. makes active enzyme. White colonies in X-gal imply the presence of recombinant DNA in the plasmid.



    Cells ready for dna uptake
    Cells ready for DNA uptake

    • Competent cells: Treat the cells with calcium chloride which makes the cell membranes more permeable to DNA. This technique succeeds with species that aren't naturally competent e.g. E. coli.

    • Electroporation - alternate method


    Finding the proper orientation of clone
    Finding the proper orientation of clone

    • Insert can go in both directions

    • How to determine correct orientation

    • Perform restriction digests using enzymes outside the cloning fragment

    • Add total fragments up

    • Must add up to right size


    Link to orientation
    Link to Orientation

    • http://homepages.strath.ac.uk/%7Edfs99109/BB211/RDTSampleAnswers.html


    Finding the right clone
    Finding the right Clone

    • Hybridization (see overhead as well)


    Genomic library
    Genomic library

    • Source of DNA to clone

    • all the cells in your body have identical DNA

    • problem with this method is introns



    Cdna libraries alternate source complimentary dna library
    cDNA libraries: alternate source(complimentary DNA library)

    • Made from RNA by reverse transcription (reverse transcriptase is enzyme)

    • RNA made into double stranded DNA

    • comes from tissue that expresses gene(s) of interest

    • no introns

    • source abundant in message

    • difficult to work with- RNA degrades more rapidly than DNA





    Alternate cloning tool pcr
    Alternate cloning tool - PCR

    • Polymerase chain reaction

    • amplification of small DNA quantities

    • clone from genomic or cDNA source

    • thermostable polymerase - heat to separate DNA strands








    Required components of pcr
    Required Components of PCR

    • DNA template DNA

    • thermocycler (or water baths)

    • pool of free dNTPs

    • Taq (or other heat-stable) DNA polymerase

    • Primers - annealed at appropriate temperatures


    Conditions for pcr
    Conditions for PCR

    • Denature: 94C to 100C , 1 minute

    • For anneal temperature, 2C for every A and T, 4 C for every C and G. 1minute - 2 minutes - GO 3-5 DEGREES BELOW THAT TEMPERATURE

    • Extension: 72 C for 2 minutes

    • Do this 30 cycles

    • machine programmable


    Problem
    Problem

    • What is the annealing temperature for the following primer (a 21 mer)?:

      AAGCTTGTCCAGAATTTCGGC


    Solution
    Solution

    • 11 A/T X 2 = 22

    • 10 C/G X 4 = 40

    • 22 + 40 + 62

    • Go a few degrees below that number, so you would anneal at about 58C


    Applications of recombinant dna
    Applications of recombinant DNA

    • Diagnosis of genes by RFLP (restriction fragment length polymorphisms)

    • Example sickle cell anemia


    Rflp restriction fragment length polymorphism
    RFLPrestriction fragment length polymorphism

    converts a GAG codon (for Glu) to a GTG codon for Val

    abolishes a sequence (CTGAGG, which spans codons 5, 6, and 7) recognized and cut by one of the restriction enzymes.


    Other diseases identified by rflp
    Other diseases identified by RFLP

    • Cystic fibrosis

    • Huntington’s disease

    • Loss (or gain) of restriction enzyme sites when amino acid change in middle of codon, and thus, protein


    How do you know sequence of dna
    How do you know sequence of DNA?

    • Sanger sequencing - named after Fred Sanger

    • utilizes 2',3'-dideoxynucleotide triphospates (ddNTPs), molecules that differ from deoxynucleotides by the having a hydrogen atom attached to the 3' carbon rather than an OH group. (see upcoming figure)


    Sanger dideoxysequencing sequencing
    Sanger (dideoxysequencing) sequencing

    • Need polymerase

    • dNTPs

    • ddNTPs

    • primer

    • DNA template




    Cellular expression systems
    Cellular expression systems

    • Expression systems are based on the insertion of a gene into a host cell for its translation and expression into protein

    • types of available systems

    • .


    Bacteria
    Bacteria

    • Advantages:

      • short generation time

      • simple physiology

      • large yield of some proteins

    • Disadvantages:

      • no post-translational modifications - glycosylation, phosphorylation

      • degradation of proteins

      • misfolded proteins


    Yeast
    Yeast

    • Advantages:

      • can perform post-translational modifications

      • secrete proteins in media- easy to isolate from there

    • Disadvantages:

      • active proteases


    Insect cells
    Insect cells

    • Advantages:

      • high expression level

      • correct folding

      • correct post-translational modification

    • Disadvantages:

      • slow generation time

      • costly- media and cells

      • finicky


    Mammalian cells
    Mammalian cells

    • Advantages:

      • cellular machinery same as gene of interest

      • folding, post-trans. Correct

      • amino acid bias the same

    • Disadvantages:

      • expresses endogenous protein, need to find correct cell line (by trial and error)


    Mammalian expression vectors
    Mammalian Expression vectors

    • Transient transfection - put into cells and protein expressed for a short period of time- usually 24 to 48 hours

    • stable tranfection - integrated into genome- expression carried on indefinitely (need to select)

    • expression vector allows for translation as well


    Introducing dna into cells
    Introducing DNA into cells

    DEAE dextran - an inert carbohydrate polymer (dextran) coupled to a positively charged chemical group (diethylaminoethyl -DEAE). DNA probably sticks to DEAE-dextran via its negatively charged phosphate groups.

    Calcium phosphate - forms an insoluble precipitate with DNA. It was discovered that cells efficiently take up this precipitate. More efficient than DEAE dextran or many cell types and can be used for both transient and stable transfection. Not suitable for cells which grow in suspension culture.


    Introducing dna into cells1
    Introducing DNA into Cells

    Electroporation - Cells are concentrated, mixed with the DNA and placed in a small chamber with electrodes connected to a specialised power supply. A brief electric pulse is applied, which is thought to ‘punch holes’ in the cell membrane, enabling the cell to take up DNA.

    Lipofection - (liposome-mediated gene transfer) several lipid- based methods have been developed in which DNA is encapsulated by synthetic lipid bilayers which resemble cell membranes. Liposomes are essentially spheres of synthetic membrane filled with DNA. These fuse spontaneously with cell membranes, releasing their contents into the cytoplasm.


    Introducing dna into cells2
    Introducing DNA into cells

    • Microinjection - The most efficient artificial means of getting DNA into cells. DNA is injected into the nucleus using a microelectrode needle. Very tedious method because each and every cell has to be injected individually. There are now computer-based systems which will assist in the process.


    Creating a fusion protein
    Creating a fusion protein

    • Gene products are “fused” together, produced as a single polypeptide

    • can then use a tag sequence to help isolate that protein

    • can purify over a column and get rid of tag by cleavage (cutting)


    Technique of cell culture
    Technique of Cell Culture

    • Follow handout – 4 pages

    • How did Tissue Culture develop

    • What is Tissue Culture

    • How is T.C. performed

    • What can go wrong

    • References


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