1. Biotechnology
Ch 20

2. 20.1 DNA cloning Making copies of segments of DNA
Gene cloning – making multiple copies of a gene
Why?
To make many copies of a gene (amplification)
To produce a protein product

3. Cloning & bacteria Plasmids are frequently used in cloning genes
Gene of interest is inserted into plasmid:
Recombinant DNA – DNA from 2 different sources
Plasmid is inserted into bacteria, bacteria divide, producing copies of genes

4. Recombinant DNA DNA from two sources is combined

5. Cloning a Gene 1. Isolate vector and gene of interest
Determine vector – molecule that will carry foreign DNA, and gene of interest
Vector may have particular genes to aid in recognition of of cell clones
vector - bacterial plasmid
Has ampR – ampicillin resistance gene
Has lacZ gene – catalyzes hydrolysis of lactose sugar – at restriction site, so the enzyme cuts in middle of gene
Gene - human gene of interest

6. Restriction Enzymes Protects bacteria by cutting up foreign DNA
Work on specific sequences of DNA, usually symmetrical
Result in “sticky ends”

7. 2. Insert gene into vector
The same restriction enzyme is used to digest the plasmid (only one recognition site), and human DNA
Result- human DNA is cut into many fragments – one is the correct one.
Cut plasmids and DNA fragments are mixed together. Sticky ends join through complementary base pairing. DNA ligase is used to form phosphodiester bonds to join DNA molecules.

8. Making recombinant DNA

9. 3. Introduce cloning vector into cells
Bacterial cells take in recombinant plasmids through transformation, taking in DNA from surrounding solution

10. 4. Cloning of cells
Bacterial cells are plated out onto nutrient medium with ampicillin and X-gal sugar added
Need to determine which bacterial cells contain recombinant plasmids
Only bacteria with recomb. plasmids will grow on medium with ampicillin, because of ampR gene
Bacteria with the intact lacZ gene turn blue with hydrolysis of X-gal, but bacteria with recomb plasmids cannot process X-gal sugar, so are white

12. Cloning a gene - video
hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::5 35::/sites/dl/free/ /120078/micro10.sw f::Steps%20in%20Cloning%20a%20Gene

13. 5. Identify cell clones with gene of interest
Need to find bacteria with plasmids that contain gene of interest, vs. other human DNA fragments
Use nucleic acid probe – short strand of DNA or RNA that is complementary to part of gene of interest
DNA is denatured, and then radioactive or fluorescent probe is added

15. Genomic Libraries A collection of many clones
A complete set of plasmid-containing cell clones
When no single gene is target, genome broken into fragments, each gets recombined into a plasmid
Bacterial artificial chromosome – larger than plasmids, hold more genes

16. Complementary DNA - cDNA
Eukaryotic DNA from its original source includes introns
To get around this problem, start with a fully processed mRNA strand
Use reverse transcriptase to synthesize double stranded DNA
Can build a cDNA library

17. Nucleic Acid Hybridization
Can be used to label particular bands of DNA
Synthesized radioactively labeled RNA hydrogen bonds with target complementary DNA

18. Cloning & expressing eukaryotic genes
Problems due to differences in how prokaryotic & eukaryotic cells express genes
Promoter- use an expression vector with a promoter sequence upstream of insertion site, so host cell recognizes it and will express gene that follows
Introns – find processed mRNA, use reverse transcriptase to make complementary DNA (cDNA) that can be used in bacteria

19. Yeast – hosts for eukaryotic cloning
Advantages:
Single- celled fungi, easy to grow
Have plasmids (unusual for eukaryotes)
Eukaryotic host cells can modify proteins after translation, bacteria can’t do this

20. PCR – Polymerase Chain Reaction
Making copies of DNA
Uses heating & cooling cycles to:
1) denature – separate DNA strands
2) anneal - bind primers at ends
3) extension -synthesize DNA with DNA polymerase

21. 20.2 DNA Technology – analyzing genes
Gel Electrophoresis:
Use electricity to separate DNA fragments in an agarose gel
DNA is negatively charged
Longer molecules travel slower than shorter molecules

22. Restriction fragment analysis
DNA can be digested with restriction enzymes, and then analyzed

23. Genome mapping DNA sequencing – dideoxy chain termination:
Sequencing by synthesis:
Human genome sequencing – shot gun sequencing:

24. Analyzing Gene Expression
Transcription is a measure of gene expression
Use probes to measure amt of mRNA present, as a way to quantify gene expression
DNA microarray assays – a grid of single strand DNA fragments, get tested for hybridization with cDNA molecules
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25. FISH
Fluorescent in situ hybridization - to determine which cells are expressing certain genes U

26. Gene function In vitro mutagenesis
Add inactive genes with a marker (mutated genes), put the gene back into the cell so it “knocks out” the normal functioning gene
RNAi – use of synthetic double strand mRNA to breakdown mRNA or block translation; acts to knock out certain genes

27. Knock out mice – Mario Capecchi
scientist-on-knockout-mice
RNAi – use of double stranded mRNA molecules to “knock out” genes

28. SNP – Single nucleotide polymorphism
A single base pair site where variation is found
Used as genetic markers for particular diseases
Find common genetic marker for people who are affected with a disease
Study nearby region of DNA to look for genes involved in disease

29. Cloning – organisms from single cells
Plants – cells from adult plants incubated in medium can grow into normal adult plants
Animals – nuclear transplantation (i.e. Dolly)
The adult cells need to be dedifferentiated
Nucleus from a differentiated adult cell is transplanted into a egg cell with the nucleus removed
Problems – defects: premature death, obesity, liver failure
Problems appear to be due to chromatin methylation issues

30. Stem Cells
Stem cells are unspecialized and can differentiate into specialized cells.
In a stem cell, DNA is arranged loosely.
In a differentiated cell, genes not needed are shut down

31. Embryonic stem cells – from embryos in the blastula stage
Can reproduce indefinitely, can differentiate into many different cell types – pluripotent
Why are they valuable?
have the potential to supply cells to repair damaged or diseased organs
Adult stem cells – can differentiate, but not as widely as embryonic stem cells

32. Induced Pluripotency -
Shinya Yamanaka took adult fibroblast cells (connective tissue cells)
Reprogrammed the cells to become pluripotent- to being capable of differentiating into different cell types (like stem cells)
Reprogrammed cells with master transcription factors
Yamanaka won the Nobel Prize in Medicine 2012

33. Current research with pluripotency
Problems with traditional genetics approach due to cancer causing genes
Use of small compounds to mimic transcription factors
Use of drug like chemicals to enhance reprogramming

34. Another way to create pluripotent cells
Haruko Obokata from Riken Center for Developmental Biology, Kobe, Japan
Stimulus-triggered acquisition of pluripotency (STAP)
Took lymphocytes from mice, bathing them in acid solution for about 30 minutes.
Cultivated the cells by adding a special protein.
In two to three days, the process had transformed the cells into pluripotent cells. They developed into nerve and muscle cells.

35. Mouse embryo injected with pluripotent cells (labeled with fluorescent protein)

36. Applications of DNA Technology
Diagnosis of diseases
Gene Therapy
Production of proteins for market
Other pharmaceutical products
Forensic evidence
Environmental Cleanup
Agricultural applications