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Human protein production. Transgenic organisms. Forensic analysis. Human genome project. RFLP analysis. DNA Technology and Genetic Engineering. DNA Technology and Genetic Engineering (making changes in DNA). Nucleus. Chromosome. 1: Production of human proteins 2: To identify people - PowerPoint PPT Presentation
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+
DNA Technology and Genetic Engineering
Human proteinproduction
RFLP analysisHuman genome
project
Transgenicorganisms
Forensic analysis
+ DNA Technology and Genetic Engineering (making changes in DNA)1: Production of human proteins
2: To identify people
3: To identify human diseases
4: To identify all human genes
5: To genetically engineer foodSupercoils
Coils
Nucleosomes
Histones
Nucleus Chromosome
DNA
Cell
+What is Dwarfism?
Dwarfism is a recessive disease that causes adults to be no more than 4 feet tall
Little people produce little or no growth hormone, which is made by the pituitary
Researchers studied families with dwarfism and found that people with dwarfism have defective copies of the gene, GH1
+Early attempts to treat Dwarfism
Attempts to inject growth hormone from pigs (a strategy that worked previously for insulin) did not work – only GH from humans would work (until 1982 source from human cadavers and up to 20,000 pituitaries were needed!)
Some of these pituitaries were contaminated with prions, which cause degenerative brain disorders
+Solution: Use recombinant DNA to produce GH in bacteria!
Recombinant DNA = DNA that results from combining DNA from different sources ex. mouse + human DNA human + bacterial DNA
+Plasmidisolated
1Bacterium
Bacterialchromosome
Plasmid
2DNAisolated
Cell containing geneof interest
DNAGene ofinterest
3 Gene inserted into plasmid
Recombinant DNA(plasmid)
4 Plasmid put intobacterial cell
Recombinantbacterium
5
Copies of gene Copies of protein
Clones of cellGene for pestresistanceinserted intoplants
Gene used to alter bacteriafor cleaning up toxic waste
Protein used to dissolve bloodclots in heart attack therapy
Protein used to make snow format highertemperature
Cell multiplies withgene of interest
Recombinant DNA Overview
+How Do You Make Recombinant DNA?
How do you make recombinant DNA? We need A) To isolate genes with restriction enzymes B) A vector C) To combine the genes and the vector
What do we do with it? D) Transfer the recombinant DNA to the host E) Find the gene of interest (human growth
hormone)
+ A) Isolate genes with restriction enzymes (DNA scissors)
Occur naturally in bacteria – why? Bacteriophages
infect bacteria Cut up foreign DNA
Hundreds are purified and available commercially
Recognize and cut at specific base sequences in DNA (usually 4-8 bases long)
+ Products generated by restriction enzymes
A) Sticky-end cutters
Enzyme Recognition site DNA after cuts
B) Blunt-end cutters
Enzyme Recognition site DNA after cuts
5’...G3’...CTTAA
AATTC...3’ G...5’
5’...CCC3’...GGG
GGG...3’CCC...5’
5’...CCCGGG...3’3’...GGGCCC...5’
SmaI
EcoRI5’...GAATTC...3’3’...CTTAAG...5’
+ A) Isolate genes with restriction enzymes (DNA scissors)
Take genomic DNA (in this case, human cells) and cut it with a particular restriction enzyme
MANY restriction fragments formed
ALL parts of the DNA are cut whenever there is a restriction site
Now what? Put these fragments in a vector
+ B) Vectors
Vector = something to carry the gene of interest into the host (i.e. bacteria) A. Mechanical – micropipettes or gene
guns B. Biological – virus or plasmid
Plasmid – additional, free-floating ring of DNA found only in bacteria Can replicate within a cell (has
origin of replication) Has antibiotic resistance gene
Cut both the plasmid and gene with the same restriction enzyme and their ends will hydrogen bond = gene splicing
+
C) Combine the gene of interest and the vector by sealing ends with DNA ligase… now you have made recombinant DNA
DNA1
Restriction enzymerecognition sequence
Restriction enzymecuts the DNA intofragments
Sticky end
2
3
4
5
Restriction enzymecuts the DNA intofragments
Addition of a DNAfragment fromanother source
Two (or more)fragments sticktogether bybase-pairing
DNA ligasepastes the strand
Recombinant DNA molecule
+D) Transfer the Recombinant DNA to the Host (Transformation)Recombinant DNA is transferred to a host cell.
Can use heat-shocking or electricity to get plasmid into bacteria
How do we know which bacterial cells have our plasmid? Antibiotic resistance gene! Resistance gene allows ONLY those bacteria with the
plasmid to grow in media that have an antibiotic on it
When the host cell copies its DNA (replicates), it also makes a copy of the plasmid. Results in clones, which are genetically identical copies.
+D) Transfer the Recombinant DNA to the Host
We use bacteria because they reproduce very quickly and have all the protein synthesis machinery (enzymes, ribosomes) Insulin for Type I diabeticsBlood factor VIII-hemophilia (clotting
factor)Antigens for vaccines (Hep B, flu,
meningitis)Cutting chromosomes in order to study
individual pieces
+E) Find the gene of interest
Each clone consists of identical cells containing one fragment (of many) of human DNA
The collection of all the cloned DNA fragments is known as a genomic library Each fragment represents
a “book” that is “shelved” in plasmids inside bacterial cells
Thus, it is a library of all the organism’s genes Figure 12.6
Genome cut up with restriction enzyme
Recombinantplasmid
OR
Recombinantphage DNA
Bacterialclone
Phageclone
Phagelibrary
Plasmidlibrary
+ E) *Side note – libraries can also be made from cDNA
The enzyme reverse transcriptase can be used to make a smaller library, called a cDNA library
This contains only the genes that are expressed (transcribed) by a specific cell type (rather than ALL the genes found in an organism)
These genes can then be digested and placed in vectors
Why is this useful? Bacterial mRNA does not have introns – doesn’t have
the machinery to splice eukaryotic genes Can help a researcher study the genes responsible for
specialized functions of a certain cell type
+
Transcription1
CELL NUCLEUS
DNA ofeukaryoticgene
RNAtranscript
mRNA
Exon Intron Exon Intron Exon
TEST TUBE
Reverse transcriptase
cDNA strand
cDNA of gene(no introns)
RNA splicing(removes introns)
2
Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand
3
Breakdown of RNA4
Synthesis of secondDNA strand
5
+E) Find the gene of interest
How do we find the right “shelf” in the library?
If we know at least part of the DNA sequence, we can create a nucleic acid probe
Probe = radioactively labeled single-stranded DNA piece that can base pair with a particular sequence
Ex: ATCCGA
The probe is mixed with clones that have been heated or treated with a chemical to separate the DNA strands
The probe will “tag” the correct “shelf” or bacterial clone that contains the gene of interest
+E) Find the gene of interest
Radioactiveprobe (DNA)
Mix with single-stranded DNA fromvarious bacterial(or phage) clones
Single-strandedDNA
Base pairingindicates thegene of interest
+E) Find the gene of interest
Bacterial colonies containingcloned segments of foreign DNA
Radioactive DNA
Transfercells tofilter
1Solutioncontainingprobe
Treat cellson filter toseparateDNA strands
2 Add probeto filter
3 ProbeDNA
Gene ofinterest
Single-strandedDNA from cell
Hydrogen-bonding
Autoradiography4
Developed filmColonies of livingcells containinggene of interest
Compare autoradiographwith master plate
5
Master plate
Filterpaper
The bacterial colony can then be grown and the protein of interest can be isolated in large amounts
+E) Isolating the gene of interest
Isolate DNAfrom two sources
1E. coli
Cut both DNAs with the same
restrictionenzyme
2
Plasmid
Human cell
DNA
Gene VSticky ends
Mix the DNAs; they joinby base-pairing
3
Add DNA ligaseto bond the DNA covalently
4
Recombinant DNAplasmid Gene V
Put plasmid into bacteriumby transformation
5
Clone the bacterium6
Bacterial clone carrying manycopies of the human gene