Biology 3A Lab PCR Lab Part 2 – Analyzing your DNA using gel electrophoresis Page 1 of 7 BIO 3A Laboratory PCR Lab Part 2 - Analyzing Your DNA Using Gel Electrophoresis Objectives • To learn how to micropipet • Understanding the principle of electrophoresis • Differentiate between introns and exons Introduction What Can Genes and DNA Tell Us? It is estimated that the 23 pairs, or 46 chromosomes, of the human genome (23 from the mother and the other 23 from the father) contain approximately 30,000 to 50,000 genes. Mach chromosome contains a series of specific genes. The larger chromosomes contain more DNA, and therefore more genes, compared to the smaller chromosomes. Each of the homologous chromosomes (pairs) contain similar genes. Each gene holds the code for a particular protein. Interestingly, the 30,000 50,000 genes only comprise 5% of the total chromosomal DNA. The other 95% is non-coding DNA. This non-coding DNA is interspersed in blocks between functional segments of genes and within genes, splitting them into segments. The exact function of the non-coding(intergenic) DNA is not yet known, although it is thought that non-coding DNA allows for the accumulation of mutations and variations within organisms. When DNA is first transcribed into RNA, exons are separated from each other by introns. While the RNA is still in the nucleus, intergenic sequences, or introns, are removed from the molecule. Meanwhile the exons are spliced together to form the complete messenger RNA coding sequence for each protein. This process is called RNA splicing and is carried out by specialized enzymes called spliceosomes. Surprisingly, introns often vary in their size and sequence among individuals while exons do not. This variation is thought to be the result of the differential accumulation of mutations in DNA throughout evolution. These Imitations are silently passed on to our descendants. We do not notice mutations in our non-coding DNA because they do not affect our phenotypes. However these differences in our DNA represent the molecular basis of DNA fingerprinting used in human identification and studies in population genetics. The Target Sequence-Can You Say "Alu"? The genetic code contains small repetitive DNA elements that have become randomly inserted info the human genome over millions of years. One such repetitive element is called the "Alu sequence"' (Figure 1). This it a DNA sequence about 300 base pairs long that is repeated, one copy at a tine, almost 500,000 times throughout the human genome. The origin and function of these randomly repeated sequences is not yet known. INTRON Figure One. Location of an Alu repetitive element within an intron.Biology 3A Lab PCR Lab Part 2 – Analyzing your DNA using gel electrophoresis Page 2 of 7 Some of these Alu sequences have characteristics that make them very useful to geneticists. When present within introns of certain genes, they can either be associated with a disease or merely used to estimate relatedness among individuals. In this exercise, analysis of a single Alu repeat is used to estimate its frequency in the population and as a simple measure of molecular genetic variation with no reference to disease or relatedness among individuals. In this lab you will be hunting for an Alu element in the PV92 region of chromosome 16. This particular Alu element is dimorphic, meaning that the element is present in some individuals and not others. Some people have the insert in one copy of their 16th chromosomes (one allele), others may have the insert in both copies of their 16th chromosome, while others may not have the insert on either copy of the 16th chromosome. The presence or absence of this insert can be detected using the polymerase chain reaction followed by agarose gel electrophoresis Since you are amplifying a region of DNA contained within an intron, the region of DNA is never really used in your body. So if you don't have it, don't worry. The primers in this kit are designed to bracket the region within the PV92 region that is 641 base pairs in length if the intron does not contain the Alu insertion or 941 base pairs in length if Alu is present .This increase in size is due to the 300 base pair sequence contributed by the Alu insert. When your PCR products are electrophoresed on an agarose gel, there are three distinct outcomes that can be visualized. If both chromosomes contain Alu inserts, then each amplified PCR product will be 941 base pairs long. On a gel these will migrate at the same speed so there will be one band that corresponds to 941 base pairs. If neither chromosome contains the insert then each amplified PCR product will be 641 base pairs and they will migrate as one band that corresponds to 641 base pairs. If you have an Alu insert on one chromosome but not the other, then there will be one PCR product of 641 base pairs and one of 941 base pairs. The resulting gel will reveal two bands. Electrophoresis separates DNA fragments according to their relative size (molecular weight). DNA fragments are loaded into an agarose gel slab, which is placed into it chamber filled with a conductive liquid buffer solution. A direct current is passed between wire electrodes at each end of the chamber. DNA fragments are negatively charged, and when placed in an electric field will be drawn toward the positive pole and repelled by the negative pole. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments call move more easily than larger ones. Over a period of time smaller fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in what appear as single "bands" of DNA in the gel. In the sample gel below (Figure 2), PCR amplified bands of 941 bp and 641 bp are separated based upon their size. Figure Two. Separation of DNA bands based on size. This get depicts the electrophoretic separation of the EZ Load DNA molecular mass ruler, which contains 1,000 bp, 700 hp, 500 bp, 200 by and 100Biology 3A Lab PCR Lab Part 2 – Analyzing your DNA using gel electrophoresis Page 3 of 7 by fragments (lane 1), two homozygous (+/+) 941 bp fragments (lanes 2 and 6), three homozygous (-/-) 641 bp fragments (lanes 3, 5, and 8), and two heterozygous (+/-) 941/641 bp fragments (lanes 4 and 7). Analyzing Your DNA Using Gel Electrophoresis Materials and supplies that should be