Cloning technology has many applications: Many copies of the gene are made Protein products can be produced
What is role of: amp gene in plasmid? lacZ gene in plasmid? (see step 2 p 387) Hint: X-gal product turns blue What is the next step after you have identified the colonies containing the recombinant plasmid?
Then what? The colonies of interest are grown in large quantities so that the gene of interest can be sequenced or so that its product can be produced What about the rest of the colonies? (thousands may be produced) –These will be usually be stored and used later or shared (or sold) to other scientists (“cloning by phone”) –A collection of many bacterial clones is a genomic library
Genomic Libraries (can also have phage libraries)
a target sequence can be amplified many times quickly billions of copies after 20 cycles Three step cycle: denaturation by heating, cooling and annealing primers, extension by heat stable DNA polymerase Polymerase Chain Reaction (PCR)
Gel electrophoresis separates molecules by size and charge
RFLP Analysis can be used to detect altered forms of a gene (polymorphism)
Dideoxy Chain Termination Method for DNA sequencing 1. Denature strand of DNA 2. Primer added 3. Dideoxynucleotides and normal ones mixed 4. Newly synthesized strands have nucleotides randomly added 5. dideoxy’s have fluoresecent or radioactive tags
How can we determine if the gene is functional? What do we mean by that? Does it produce a product? Do different cells produce different products? How can we tell the difference? How can we tell normal from abnormal cells? How we tell the difference between brain cells and liver cells?
Short-tandem repeats A short tandem repeat (STR) in DNA is a class of polymorphism that occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 10 base pairs (bp) (for example, CATGCATG) and is typically in the non-coding intron region, making it “junk DNA”.
STR’s (cont) An individual inherits one marker from one parent and the other from the other parent By examining several STR loci and counting how many repeats of a specific STR sequence there are at a given locus, it is possible to create a unique genetic profile of an individual. STR analysis has become the prevalent analysis method for determining genetic profiles in forensic cases.
Comparison of three loci among 4 suspects: Who does the blood stain belong to?