DNA profiling DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles. Definitions you will need to understand : Microsatellites are di-, tri-, or tetra nucleotide tandem repeats in DNA sequences. The number of repeats is variable in populations of DNA and within the alleles of an individual.  A short tandem repeat is a microsatellite, consisting of a unit of two to thirteen nucleotides repeated hundreds of times in a row on the DNA strand. STR analysis measures the exact number of repeating units.

DNA profiling is commonly used in criminal investigations (forensics) and to settle paternity disputes The procedure involved is common for both: A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR Satellite DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments Fragment length will differ between individuals due to the variable length of their short tandem repeats The fragments are separated using gel electrophoresis and the resulting profiles are compared

PCR (Polymerase Chain Reaction) It is in vitro (in the lab) amplification (making more copies) of a specific segment of DNA using a thermostable enzyme (an enzyme not affected by heat). It was invented in 1985 by Cary Mullis The PCR process makes millions of copies of DNA in just a few hours. It is a replication reaction, which uses reagents very similar to what is needed for DNA replication inside a cell. Each strand serves as a template for synthesis of its complementary strand. The analysis of DNA is used in: (Making lots of DNA for sequencing) the Human Genome Project, Producing numerous copies of genes for genetic engineering paternity testing, Finding and analyzing DNA from very small samples for use in forensics diagnosis of genetic disorders the detection of infection. Detecting the presence of disease-causing microbes in human samples

Requirements for PCR DNA – the original strand of DNA which needs amplified. Complimentary primers – primers are short complementary sequences of RNA needed to start DNA synthesis. Thermal cycler – equipment that varies the temperature of the reaction. The thermal cycler allows this process to be automated. The reaction mixture is added, and then repeated cycles of heating and cooling cause the DNA to be continually denatured and replicated. Heat-tolerant polymerase – an enzyme which will add nucleotides to the growing strand and which is not denatured by the high temperatures used in the reaction. Supply of nucleotides – to synthesize the new strands of DNA.

The PCR process DNA heated - to denature the DNA and separate the two strands. DNA cooled - in preparation for adding primers. Complimentary primers added - which are complementary to the target sequences at the two ends of the region to be amplified. Heat-tolerant DNA polymerase added - which replicates the region of DNA to be amplified. Two strands are formed. (Taq polymerase) Repeated cycles are carried out - which amplify this region of DNA from a single strand to millions of copies.

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NB: Taq polymerase is an enzyme extracted from the bacterium, Thermus aquaticus. This bacterium lives in hot springs, thus it can withstand high temperatures. During PCR, the sample is heated to 90C (for 30 sec) to denature the DNA (separate the 2 strands). Then the sample is cooled down to 50C (for 1 min) to allow the 2 primers to anneal to each DNA strand. Finally, the sample is heated to 72C (for 2 min), which is the optimal temperature for Taq polymerase to add on nucleotides after each primer. This cycle of denaturing, annealing and extending is repeated 30-40 times. For each cycle, the targeted gene is doubled in number.   1 --> 2 --> 4 --> 8 --> 16 --> 32 --> 64 --> 128 --> 256 --> 512 --> 1024 --> 2048 --> 4096 --> 8192 --> 16,384 --> 32,768 --> 65,536 --> 131,072 --> 262,144 --> 524,288 --> 1,048,576 As shown above, after 20 cycles, over a million copies of a gene from one molecule of DNA is generated. This logarithmic process is called amplification.

NB 2: Scientists can even combine DNA from different organisms to artificially create materials such as human proteins or to give crop plants new characteristics. They can also compare different versions of the same gene to see exactly where disease-causing variations occur.

Gel electrophoresis Scientists use gel electrophoresis to separate molecules based on their size and electrical charge. Gel electrophoresis can separate fragments of DNA that were cut with restriction enzymes, creating a visual map of fragment size that’s easy to interpret. Or scientists may use gel electrophoresis to separate a protein they want to study from other proteins in a cell One of the advantages of gel electrophoresis is that scientists can separate several samples side by side so they can compare them. The comparison of separated DNA molecules is the basic method behind the DNA fingerprints that forensic scientists use to compare samples from crime scenes with those of suspects.

This technique separates fragments by: charge, size (molecular weight) and shape. SOME TERMS TO HELP YOU UNDERSTAND Agarose is a polysaccharide that can be used to form a gel to separate molecules based on size. The rate at which DNA fragments can slip through the pores in this gel is based on size. Small DNA fragments wiggle through the pores in the agarose gel faster than longer fragments. A buffer is a solution that can resist pH change upon the addition of an acidic or basic components. It is able to neutralize small amounts of added acid or base, thus maintaining the pH of the solution relatively stable.

Gel Electrophoresis Process: First, an agarose gel is made with slots in it called wells. The DNA sample is dispensed in the slots A buffer solution is placed in the apparatus, and an electric current is run through the gel. DNA molecules are negatively charged due to the phosphates in its backbone, and when placed in an electric field starting at the negative (black) electrode, it will migrate towards the positive (red) electrode. The smaller DNA molecules pass more easily (due to less friction) and migrate faster through the gel than larger size fragments. Linear molecules also migrate faster through a gel, compared to globular forms. DNA fragments are stained and viewed under ultra violet light, and a brightly colored band indicates the presence of DNA