Question

nomal No Spac... Heading 1 Paragraph JULLL HDBl AaBbCcDAoBbced Heading 2 Title Subtitle Subtle Em Styles 1-What RFLP stand for and what RFLP analysis test for? 2-Why do you add sample loading buffer to DNA sample prior to loading sample into agarose gel. 3 In cheek DNA extraction procedure, name two steps that helped to break (lysis) the cheek cells. 4. Would a shorter DNA fragment move faster or slower through agarose gel, why.

Answer 1

1). restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence.The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.....  

RFLP analysis was an important early tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing....

2).DNA loading buffers are used for loading DNA samples onto agarose or SDS DNA gels for gel electrophoresis. DNA loading buffers contains a coloured dye and a density agent. The density agent serves to enhance the density of the DNA sample allowing the DNA to sink into the bottom of the well. The dye adds visibility to the DNA sample and also serves as a tracking dye allowing the user to monitor the DNA migration during electrophoresis....

3). Cell lysis by :-  
•Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this. First, mechanical disruption breaks open the cells. This can be done with a tissue homogenizer (like a small blender), with a mortar and pestle, or by cutting the tissue into small pieces. Mechanical disruption is particularly important when using plant cells because they have a tough cell wall. Second, lysis uses detergents and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins.

Add 2 ml of lysis buffer to the test tube...

Cell Lysis Buffer
• 50 mM Tris pH 8.0
• Buffering for DNA stability and optimal enzyme activity
• 1 % Sodium dodecyl sulfate (SDS)
• 1 mM Ethylenediaminetetraacetic acid

SDS Disrupts Cell Membranes

SDS Disrupts Cell Membranes SDS micelle Lipid bilayer Mixed micelle A concentration of 0.3% - 1% SDS is sufficient to disrupt the membranes of buccal cells

EDTA Inhibits Enzymes such as DNase I....

4).DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones....Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel.

Smaller molecules travel faster than larger molecules in gel, and double-stranded DNA moves at a rate that is inversely proportional to the logarithm of the number of base pairs. This relationship however breaks down with very large DNA fragments, and separation of very large DNA fragments requires the use of pulsed field gel electrophoresis (PFGE), which applies alternating current from two different directions and the large DNA fragments are separated as they reorient themselves with the changing current.[12]

For standard agarose gel electrophoresis, larger molecules are resolved better using a low concentration gel while smaller molecules separate better at high concentration gel. High concentrations gel however requires longer run times (sometimes days...