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Highlights of DNA Technology. Cloning technology has many applications : Many copies of the gene are made Protein products can be produced. Restriction enzymes are used to make recombinant DNA. What is role of: amp gene in plasmid? lac Z gene in plasmid? (see step 2 p 387)
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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
Polymerase Chain Reaction (PCR) • 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
“Sometimes a good idea comes to you when you are not looking for it. Through an improbable combination of coincidences, naivete and lucky mistakes, such a revelation came to me one Friday night in April, 1983, as I gripped the steering wheel of my car and snaked along a moonlit mountain road into northern California's redwood country. That was how I stumbled across a process that could make unlimited numbers of copies of genes, a process now known as the polymerase chain reaction (PCR).” (Kary B. Mullis, “The Unusual Origin of the Polymerase Chain Reaction,” Scientific American, 1990. www.nobel.se/chemistry/educational/poster/1993/
Southern Blotting of DNA Fragments How would you interpret lane III?
RFLP Analysis can be used to detect altered forms of a gene (polymorphism)
RFLP or “rif lip” • Different alleles have different DNA sequences. These differing sequences can be found using restriction fragment length polymorphisms • The difference in banding patterns after electrophoresis allows for diagnosis of the disease, or even a carrier of a disease
Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination. • An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.
Paternity • DNA was extracted from white blood cells from all three individuals • Is Jack the father of Jill’s child, Payle?
Does this individual carry any alleles for Cystic Fibrosis? • RFLPs are known for CF and so it would be easy to determine if a person were homozygous wild-type (wt), heterozygous "carrier", or homozygous disease alleles and thus have CF. For couples expecting a child, it would be simple to test both parents and make a prediction about the eventual disease status of their fetus. For example, if both parents were homozygous wt, then all of their children would also be homozygous wt:
Disease status • However, if both parents were heterozygous, they could have children with any of the three genotypes, though heterozygous children would be twice as likely as either of the homozygous genotypes. With increasing genomic sequence information, increasing numbers of genetic disease can be predicted from RFLP analyses.
DNA Sequencing • Human Genome Project – remember from last year?
Dideoxy Chain Termination Method for DNA sequencing 1. Denature strand of DNA 2. Add primer 3. Mix dideoxynucleotides and normal nucleotides 4. Newly synthesized strands have nucleotides randomly added 5. Dideoxy’s have fluoresecent or radioactive tags 6. Where will the shortest strands be in the gel? What does that tell you?
Computer analyzes fluoresence intensity and plots as peak height
This approach requires that you know the order of the segments. This method is very labor intensive; the advantage is that each segment only needs to be sequenced once.
In shot-gun sequencing random clones are sequenced Computer analysis of the sequences determines where the sequences overlap. With repeated sequencing enough overlap will allow the segments to be arranged in order
Additional info on this method • Figure 4. Shotgun Sequencing. A relatively crude method of sequencing, shotgun sequencing does not produce a physical map of the source clone first. Instead, the source clone is fragmented, producing a random mixture, and a random sub-clone (i.e. an unordered sequencing clone of blue, yellow, black, red or green) is selected for sequencing by the Sanger method. To ensure that that the whole source clone has been sequenced, this stretch of DNA must be sequenced numerous times (represented by multiples of a single coloured sub-clone) to produce an ordered overlapping sequence. Gaps in this process will occur where a sub-clone is not fully sequenced (blue coloured sub-clone). Source:http://www.scq.ubc.ca/genome-projects-uncovering-the-blueprints-of-biology/
How can we determine if a gene is functional?What do we mean by that? • Is it expressed, i.e., does it produce a product? (RNA or a protein) • 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?
Microarrays • Uses reverse transcriptase • Uses cDNA • Many genes present on small chip (can be 1000’s) • Measure the level of fluoresence on each point on array • Degree of fluoresence increases with more sequence complementarity
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?
