Part 2: RFLP

What is RFLP?

Restriction Fragment Length Polymorphism (RFLP) is a technique used to analyze variations in DNA sequences between individuals determined by restriction enzyme cleavage patterns. They can be used to identify unknown DNA samples because they will separate by size when in an electric field.

Restriction: What does it refer to?

• where are the from? what is their function?

Refers to restriction enzymes

Are enzymes that are produced and have been isolated from bacteria that cut DNA at very specific sequences, known as recognition sites

Fragment: What does it refer to?

when restriction enzymes cut a long strand of DNA they break into fragments

Length: What does this refer to?

because every persons DNA sequence is slightly different, the restriction enzymes will cut one persons DNA in different places than others, resulting in fragments of varying lengths

Polymorphism: What does this refer to?

variations in DNA sequences due to the points mutations that are constantly happening, producing different fragments that make each persons DNA a unique profile (except identical twins)

R: Recognition Sites/Sequences

each enzyme has its own unique recognition sites

List the steps involved in DNA fingerprinting from extraction of DNA through autoraidiography.

A. extraction of DNA from a sample such as blood, skin, semen or hair.

B. purification and amplification of the DNA.

C. digestion of the DNA into different size fragments with restriction enzymes.

D. agarose gel electrophoresis to separate fragments according to size.

E. denaturation of DNA to separate the strands.

F. Southern Blot test to transfer DNA to a membrane

G. addition of radioactive probe to hybridize complimentary DNA so that results can be exposed to X-ray and detected by audioradiography.

C. digestion of the DNA into different size fragments with restriction enzymes.

restriction enzymes cut fragments at very specific recognition sites

Explain: D. agarose gel electrophoresis to separate fragments according to size.

DNA fragments are placed into agarose gel (polymer), and an electric current is applied. Because DNA has a negative charge, due to their phosphate groups it will move towards the positive electrode.

small fragments wiggle through the gel faster than the larger fragments, effectively separating them by size

Polymorphisms in RFLP analysis

• what are the different genomic variations among individuals?

• how do they impact recognition sites?

• Single nucleotide polymorphisms (SNP)

• Copy number variations (CNV)

• can destroy,, create, or move these recognition sites

Polymorphisms: Single nucleotide polymorphisms (SNP)

single nucleotide substitution

Polymorphisms: Copy number variations (CNV)

DNA has non coding regions with repetitive sequences

Explain the significance of repeated DNA sequences.

There are sets of randomly arranged repeated DNA sequences, which vary in number from one individual to another. These sequences can occur anywhere between genes and constitute a large percentage of the human genome. It has been suggested that these sequences have been inserted possibly by viruses. As far as we can tell, they have no function. Thus, as we cut up these sections, each person will have a distinct size and number of these sequences and a pattern unique to each individual will result.

Who are the only individuals possessing the same DNA fingerprints?

Identical twins are the only naturally occurring individuals that have the same DNA fingerprints. However, modern technology has advanced far enough where it is theoretically possible to clone humans, which would, by definition, have to same DNA

5. What type of human cells can be utilized for this technique?

DNA is found in blood (WBC), semen, skin and hair (roots) and can be utilized for DNA fingerprinting. Theoretically, any cell with a nucleus (and therefore, DNA) can be used.

What are sticky ends?

are overhanging portions of DNA that can bond with complementary sequences.

Enzyme: BamHI

• what is its recognition sequence?

• what type of ends does it form?

recognizes the sequence 'GGATCC’ and cuts between GG

creates sticky ends

Enzyme: EcoRI

• what is its recognition sequence?

• what type of ends does it form?

GAATTC and cuts between GA

sticky ends

Enzyme: HaeIII

• what is its recognition sequence?

• what type of ends does it form?

GGCC, cuts between GC

forms blunt ends

Enzyme: HindIII

• what is its recognition sequence?

• what type of ends does it form?

AAGCTT, cuts between AA

forms sticky ends