Presentation on theme: "DNA fingerprinting Every human carries a unique set of genes (except twins!) The order of the base pairs in the sequence of every human varies In a single."— Presentation transcript:
DNA fingerprinting Every human carries a unique set of genes (except twins!) The order of the base pairs in the sequence of every human varies In a single human the DNA sequence in every cell is the same DNA fingerprinting is based on the two facts: there is uniform DNA in an individual there is genetic variability between individuals It is not necessary to catalogue every base pair to arrive at a unique pattern. The original DNA fingerprinting detected unique DNA segments in a population known as restriction fragment length polymorphisms (RFLPs).
A Polymorphism is a sequence of DNA that has many possible alternatives e.g. different numbers of repeats GC GC GC The polymorphic sequences tested in fingerprinting generally come from areas that do not code. i.e. introns (non coding sequences). The name RFLP is derived from the fact that when DNA, extracted from the cells of an individual, is cut with a restriction enzyme, it produces a unique pattern of bands when the cut DNA is subjected to electrophoresis. Polymorphic differences in the sequence from individual to individual give rise to alterations in the sizes of the fragments cut by restriction enzymes. The pattern of restricted DNA fragments (different lengths of DNA) that appears after electrophoresis can be thought of as a bar code. The bands from cells of one individual can be very different from those of another. This is due to the presence of hypervariable regions in the genome.
Among these hypervariable regions are variable number tandem repeat sequences (VNTR) The number of the VNTRs can vary significantly from individual to individual In humans such sequences are often bordered by restriction endonuclease sites. The fragment sizes resulting from digestion depend on the number of copies between the restriction sites This gives rise to unique RFLP patterns.
The original procedure used to obtain a DNA fingerprint 1.Isolate genomic DNA 2.cut with restriction enzymes 3.run on a gel As humans have more than 3 billion base pairs in their genome, after electrophoresis all that can be seen is a smear because all the resulting bands overlap. To visualise a fingerprint pattern of a specific VNTR site the DNA fragments must be detected by probing and hybridisation. 4. After electrophoresis the DNA in the gel is denatured and transferred to a membrane to make a permanent record. 5.The membrane is then “probed” using a piece of sequence that is complimentary to the hypervariable region. 6.The binding of the probe is visualised using radioactivity, fluorescence, conjugated enzyme. 7.The resulting band patterns are a fingerprint. 8.The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously.
Today because we have the human DNA sequence and certain other genome sequences instead of digesting total genomic DNA and creating a permanent record on a membrane that is then probed for variable regions, several different highly variable regions are amplified directly by PCR FBI uses 22 different regions, RCMP 15 different regions, paternity tests typically use at least 7 different regions
Applications of DNA fingerprinting microbial diagnostics genetic diagnostics forensic identification paternity analysis phylogenetic identification
each person's DNA is as unique as a fingerprint. Blood from the crime scene evidence, victims blood, and blood from 2 suspects Markers and control DNA to make sure the the probes are working
Suspect 2 can be eliminated Is suspect 1 guilty Can not be certain. The best we can do is to estimate the probability that another person, picked at random, could provide the same DNA fingerprint. The more probes you use, the more confident you can be that you have caught the right person.
Major issues with forensic data Reliability of Population Data The probability of finding a match for a particular DNA pattern is found by multiplying the probability of the separate loci in a particular reference population. The FBI has developed a database of population statistics for Caucasians, Blacks, Hispanics and Asians. The density of the population must also be considered Determining a Match This becomes difficult when there are only small differences in the number of variable number tandem repeats at a particular locus. Problems are also caused because of "band shifting" which may occur with differences in the gel or excess DNA in a well. Contamination and Degradation of DNA Samples. Samples may be contaminated because of bacterial growth in the sample before it was collected. Old samples may also break down and give inadmissible results. These samples may have extra bands or be missing bands. Quality Control Practices of Labs. standards for labs are essential. Absence of standards may lead to poor test quality.
Microbial diagnostics Genome-based molecular diagnostics are used to: Detect microbes Identify microbes Determine antimicrobial susceptibilities Perform epidemiologic investigations
Genetic diagnostics Screening for the sickle-cell gene Caused by a mutation in a specific gene The only difference between the two genes is the substitution of a T for an A This converts a GAG codon (for Glu) to a GTG codon for Val and abolishes a sequence (CTGAGG) recognized and cut by one of the restriction enzymes.
When the normal gene (betaA) is digested with the enzyme and the fragments separated by electrophoresis, the probe binds to a short fragment However, the enzyme cannot cut the sickle-cell gene at this site, so the probe attaches to a much larger fragment In this example, a change of a single nucleotide produced the RFLP. This is a very common cause of RFLPs and now such polymorphisms are often referred to as single nucleotide polymorphisms or SNPs