PCR and RLFP’s

Polymerase Chain Reaction (PCR) A method of making many copies of a piece of DNA. It takes about 3 hours to make 1 Billion Copies of a single DNA molecule You need: DNA sample to amplify Many copies of 2 specific oligonucleotide primers These primers are synthetically made and are specific to the section of DNA you wish to copy. DNA nucleotide bases Enzyme: “Taq DNA polymerase” Found in geysers of Yellowstone National Park

Steps in Copying DNA A DNA molecule is placed in a small test tube Taq DNA polymerase, primers, and nucleotide bases are added.

Steps in Copying DNA The DNA is heated up-usually to around 90 degrees C. This breaks the hydrogen bonds between the two strands of DNA so they separate. The temperature is lowered to about 55 degrees C This allows the specific primers to bind to the DNA strand at the appropriate sequence. The primers act like bookends-only the DNA sequence between the primers will be copied

Copying DNA The temperature is increased again to around 70 degrees C, This allows the Taq DNA polymerase to add new bases to the separated strands The cycle is repeated many times over the next few hours to create many copies of the DNA.

PCR https://www.youtube.com/watch?v=2KoLnIwoZKU

Restriction Fragment Length Polymorphism (RFLP)

RFLP Restriction Enzymes are used to “cut” the strand of DNA at a specific nucleotide sequence. Each person has unique DNA and therefore, the location of the specific sequence will be different for each person. The result is many fragments of DNA that are all different lengths. We can use a technique called Gel Electrophoresis to separate these fragments and analyze the DNA. Uses: Genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting (who does the DNA belong to), and paternity testing https://highered.mheducation.com/sites/9834092339/student_view0/chapter17/restriction_fragment_length_polymorphisms.html

Gel Electrophoresis

What is it? A method used to separate and analyze DNA, RNA and proteins based on their size and charge. The phosphate groups are negatively charged so they will be attracted to the positive electrode

How does it work? Gel is added to a container hooked up to an electric current generator. Samples of the DNA, RNA or Protein are added to the wells at one end of the gel. The electric current is turned on. Particles are pulled through the gel in ladders based on their charge. Smaller particles move farther than large particles and are found near the bottom. Negative DNA moves toward the positive electrode. Die is used to stain the material based on what you want to see.

Results

How to Read A Gel The sample in each well will separate into well defined “lines” of DNA. Each line is called a band. Each band contains a large number of DNA fragments of the same size that have all traveled as a group to the same position. Together, all the bands are called a DNA ladder. We can compare the DNA ladder of each sample to a reference slot that contains fragments of a known length. We can also compare the different bands to each other to get a “match”