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DNA Profiling - Coggle Diagram
DNA Profiling
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Gel electrophoresis
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Samples of DNA are loaded into wells on a agarose gel that attached to two electrodes (anode and cathode).
DNA fragments are negatively charged due to the presence of phosphate groups in the polynucleotides strands and are attracted to the positive electrode
Separation of DNA fragments occurs due to the molecules of DNA migrating through the agarose gel at different rates
Smaller molecules travel at a greater rate and move a greater distance from the anode while the larger molecules travel at a slower rate and move a shorter distance from the negative electrode
A dyed marker containing DNA fragments of known and varying sizes is run alongside the sample to serve as reference for size
After DNA separation has occurred, a fluorescent dye is added to the DNA. The dye bonds with DNA and causes the molecules to fluoresce under UV radiation. A DNA profile can be taken which allows an analyst to identify and analyse the different sized DNA fragment in the sample
DNA profiling
A DNA profile is a DNA-based pattern composed of a series of bands corresponding to DNA fragments of different sizes
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Used in forensic science, paternity and ancestry testung, screening for genetic disease
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The genome of an individual contains exons and introns. Introns contain repetitive sequences of nucleotides bases called short tandem repeats (STR).
STR analysis
method used to compare the short tandem repeats sequence at specific loci on the DNA from two or more samples
The data collected from STR analysis is used to construct an STR electropherogram. The data from the electropherogram can be displayed in a DNA profile table
The STR electropherogram can determine; the number of repeats in each allele at a locus, total length of the STR, if the individual is homozygous or heterozygous for the STR
DNA sequencing
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Procedure
- Four test tubes are each filled with many copies of the target DNA molecule to be sequenced
- All four dNTPs are transferred to each test tube
- A different type of ddNTPs is transferred to each test tube
- Primers and DNA polymerase are added to each of the four test tubes
- The four test tubes are transferred to a thermocycler where DNA replication / PCR occurs
- Replication is stopped whenever a ddNTPs is added instead of an dNTPs. This method produced many incomplete copies of the template DNA, each ending in a ddNTPs.
- The contents of each test tube are transferred to four separate wells in agarose gel, and the fragments in the four samples are separated using electrophoresis. The relative position of the DNA fragment reveals the sequence of the target DNA molecule
Electropherogram
A fluorescent chemical compound is attached to the ddNTPs used in DNA sequencing and both are positioned at the end of a DNA fragment
Capillary gel electrophoresis is used to separate the fluorescently labelled DNA fragments by size and this process occurs within thin capillary tubes inside a DNA sequencer
DNA fragments migrate through the capillary tubes at different rates with smaller fragments moving faster than larger fragments. All fragments pass through a beam of laser light which is absorbed and re-emitted by fluorescent molecules attached to the ddNTPs. The emitted light enter a detector.
The detector converts the emitted light into an electrical current that is analysed by computer software. The software produces a graph of the data received from a DNA sequencer, and this graph is called an electropherogram
Each peak in an electropherogram represents one of the four DNA nucleotides. The height of each peak represents the amount of light absorbed and emitted by the fluorescent molecule attached to the ddNTPs at the end of a DNA fragment. The order of the peaks represent the nucleotide sequence of the target DNA molecule
PCR
The polymerase chain reaction is a technique used to create multiple copies of a chosen segment of DNA.
Forensics: DNA in the hair follicles or blood can be amplified and this can lead to the identification or elimination of individuals
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Steps for PCR
- The temp is increased to around 95 which breaks the hydrogen bonds between the DNA strands of the target DNA
- Then the temp is decreased to around 55 so that the primers can anneal to the separated strands of target DNA through the formation of hydrogen bonds between the complementary bases
- The temp is increased to 72 which is optimum for the DNA polymerase enzyme. DNA polymerase extends the primers and create new polynucleotide strands using DNA nucleotides
DNA Extraction
- Add detergent to breakdown and dissolve cellular and nuclear membranes
- Treat cell contents with protease to destroy proteins and RNAase to destory RNA
- Place sample in centrifuge to form a pellet of all the cell debris
- Take the liquid that contains the DNA and transfer it into a clean test tube and add alcohol. This causes the DNA to form a strand which can be wind around on a glass rod