• 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

• 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

• 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

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

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

• 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

– cuts can leave blunt or sticky ends

Constructing recombinant DNA:

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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• 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 http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_ quiz_3.html

Constructing a cDNA library:

• 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

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5e/animation0412.html

A. PCR
• Makes many copies of a
DNA sample
• Uses a three step cycle:
• Heating to about 95 o
C to separate strands
• Cooling to anneal primers
• Replication using ___ https://highered.mcgrawhill.com/sites/dl/free/0072835125/126997/a nimation38.html

1. Electrophoresis
• 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 http://bcs.whfreeman.com/thelifewire
/content/chp16/1602001.html

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
– http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html

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 http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch apter15/animation_quiz_1.html

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

• 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

SNPs:

Short Tandem Repeats:

• 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
• https://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation40.html