Advanced Molecular Biological Techniques

Polymerase Chain Reaction

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Polymerase Chain Reaction

• PCR is used to create numerous copies of specific DNA fragments

• The method is closely related to the process of replication

• http://www.gene-quantification.de/block.html

Separating the Double Stranded DNA

• DNA is subjected to a temperature in the 94°C–96°C range.

• At such a high temperature, the hydrogen bonds are broken between complementary bases, and the strands separate.

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/pg2.html

Primers

• The two single strands can be used as templates to build complementary strands

• RNA primers and are used because they are easily synthesized

• The RNA primers are complementary to the target area to be copied

• one of the primers is the complement of one end of the target DNA

• the other primer is the complement of the opposite end of the opposing strand

http://www.flmnh.ufl.edu/cowries/amplify.html

Annealing the Primers

• The temperature is brought down to the 50°C–65°C range for the primers to anneal with the template DNA

http://www.cryst.bbk.ac.uk/pps97/assignments/projects/borek/Domina/page3a.html

Taq Polymerase

• Taq polymerase is a DNA polymerase so it can build complementary strands using free nucleotides that have been added

• The synthesis of the DNA strand takes place at a temperature of 72°C.

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Taq Polymerase

• Taq polymerase is isolated from Thermus aquaticus, a bacterium that lives in hot springs

• It has enzymes that can withstand high temperatures.

• DNA polymerase III denatures above 37°C, so it is not practical or efficient to use.

Repeating the cycle• Each subsequent cycle doubles the number of

double-stranded copies of the target area, resulting in an exponential increase in the number of copies of the target DNA.

http://www.madehow.com/Volume-6/DNA-Synthesis.html

Exponential increases

• After about 30 cycles, more than 1 billion copies of the targeted area will exist (230)

Isolating Target DNA• The targeted area is not completely isolated in

the first few cycles of DNA replication.• After four cycles half the fragments consist

only of target DNA and half the fragments contain flanking sequences as well

Uses of PCR

• In criminal investigations, forensic scientists can find enough DNA in a hair follicle or one cell to use as a starting point for PCR.

• HIV cannot be detected immediately by looking for antibodies, because it takes time for the body to build antibodies against it.

• With PCR, primers can be designed to complement short regions of the DNA of HIV so that the DNA can be amplified and examined for the presence of the HIV genome.

• Researchers can use PCR to determine, from fossil remains, whether or not two species are closely related.

History

• Kary Mullis first proposed the process of PCR in 1987.

• Mullis was awarded the Nobel Prize in Chemistry in 1992 for his development of PCR.

RFLP

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Restriction Fragment Length Polymorphism

• Polymorphism is any difference in DNA sequence that can be detected between individuals.

• Genomes of individuals from the same species are said to be polymorphic unless the individuals are identical twins. http://www.genetics.edu.au/factsheet/fs22

Restriction Fragment Length Polymorphism

• Polymorphism is any difference in DNA sequence that can be detected between individuals.

• Genomes of individuals from the same species are said to be polymorphic unless the individuals are identical twins.

Restriction Fragment Length Polymorphism

• Polymorphisms occur both in coding and noncoding regions.

• In coding regions, polymorphisms may be used to identify individuals with specific mutations

• In noncoding regions, variations may exist in the number of variable number tandem repeats, also known as microsatellites

Restriction Fragment LengthPolymorphism (RFLP) Analysis

• A technique in which DNA regions are digested using restriction endonuclease and subjected to radioactive complementary DNA probes to compare the differences in DNA fragment lengths between individuals

Southern Blot Technique

• http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html

Restriction Fragment Length Polymorphism Analysis

• The DNA is first digested using either one or several different restriction endonucleases.

• The digested DNA is then run on a gel• It appears as a long smear because many

fragments differing only slightly in size over a wide range are produced

Southern Blotting Technique

• The gel is subjected to a chemical that causes the DNA to be denatured.

• The single-stranded DNA is then transferred to a nylon membrane with the aid of an electric current.

• This transfer procedure is named after E.M. Southern

http://oregonstate.edu/instruction/bb492/fignumbers/Fig25D-1.html

Southern Blot Technique

• The nylon membrane containing the single-stranded DNA is immersed in a solution containing radioactive complementary nucleotide probes

• Probes may be mutations in an allele that lead to a specific disease, or a variable number tandem repeat found in a noncoding region of the organism’s DNA.

RFLP Analysis

• Complementary base pairing will occur between the probes and the DNA

• The nylon membrane is placed against X-ray film.

• When the film is developed, a pattern is detected.

RFLP Analysis

• The differences in pattern can be used to match a suspect’s DNA to the DNA found at a crime scene, or to detect a mutation that is believed to cause a genetic disorder.

http://www.bio.miami.edu/dana/250/25007_8.html