1. GENETIC ENGINEERING CHAPTER 20

2. In order to do these, scientists need the genes to study and sequence
Genetic engineering is the ability of humans to modify and manipulate DNA for:
Identification of genetic disorders
Gene therapy
Crop and food production
Tailoring medicines for the cancer
Creating recombinant medicines, vaccines
Forensics
Analyzing evolutionary relationships
In order to do these, scientists need the genes to study and sequence

3. I. DNA Cloning
Involves inserting DNA into a vector and cloning it into a cell for production of multiple copies
A. Vectors
DNA molecules that will carry foreign DNA into cells: plasmids, BACs, YACs

4. Plasmid: small circular DNA found in bacteria.
Antibiotic resistant gene allows selection of bacteria that took up plasmid
Multiple cloning site allows insertion of foreign DNA

5. Bacterial artificial chromosome (BAC): large plasmid that can carry large DNA fragments in bacterial cells

6. Yeast artificial chromosome (YAC): derived from yeast DNA and used to clone really large DNA fragments into eukaryotic cells

7. B. restriction enzymes
found naturally in bacteria to protect them from invading viruses
molecular scissors that cut DNA at specific nucleotide sequences
enzyme binds to DNA at that sequences and cuts between the sugar and phosphate on both strands

8. cuts can leave blunt or sticky ends

9. Constructing recombinant DNA:

10. II. How to clone a gene using a plasmid vector
A. Basic procedure
Cut the DNA of interest and vector with the same restriction enzyme (genomic or cDNA)
Fragments are mixed and ligase seals fragments together
Vector DNA will incorporate the fragments of source DNA in them creating recombinant plasmid
Some vectors will NOT incorporate foreign DNA. They are nonrecombinant
Cells are transformed by mixing vector with cells
Recombinant cells are selected for using agar plates with antibiotics

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12. B. DNA libraries
Contain fragmented DNA from a particular species stored in a vector ready for cloning
Genomic libraries: made from genomic DNA
cDNA libraries: made from a species’ mRNA using reverse transcriptase

13. Constructing a cDNA library:

14. C. Screening for the gene of interest
Use a radioactive probe that will only recognize gene of interest
Transfer some of the bacteria to a piece of filter paper forming a replica
Incubate filter with radioactive piece of DNA complementary to gene of interest
Make an X-ray film.
Use the X-ray film to isolate colony with gene of interest

16. III. DNA technology PCR Makes many copies of a DNA sample
Uses a three step cycle:
Heating to about 95o C to separate strands
Cooling to anneal primers
Replication using ___

17. B. Electrophoresis and Blotting
Uses a gel as a molecular sieve to separate nucleic acids or proteins based on size
A current is applied to gel that causes the charged molecules to move thru the gel forming bands based on size
Bands are visualized with some type of dye

18. Southern blot analyzes DNA Northern blot analyzes mRNA
2. Blotting
After electrophoresis, bands are transferred to a filter paper for analysis of a particular fragment (a gene fragment, mRNA, or protein)
Filter paper is incubated with a “probe” to the particular fragment of interest
Southern blot analyzes DNA
Northern blot analyzes mRNA
Western blot analyzes proteins

20. Northern blot

22. 3. DNA sequencing
Small stretches of DNA are sequenced using dideoxy chain termination method
Uses dideoxynucleotides (ddNTP) mixed with normal ones
Each type of ddNTP has a different fluorescent tag
 DNA sequence is read

23. Manual sequencing done in 1980s and 1990s
Automated sequencing done now:

24. IV. The human genome is polymorphic
Genetically, humans are 99% identical but:
Alleles have small nucleotide differences
Different individuals have different numbers of short tandem repeats: a sequence of 2-5 nucleotides repeated over
This is restriction fragment length polymorphism

25. SNPs:

26. Short Tandem Repeats:

27. A. Creating genetic profiles using STR analysis

28. There are many regions on chromosomes where STRs exist.
The number of tandem repeats in each region varies from individual
By using PCR on the STR’s of an individual, you can create a genetic profile

32. Cloning an organism: