M W F; 10:10 - 11:00 pm
Molecular Biology

Douglas W. Smith

York 2722
BIMM 100

5254 Muir Biology Building

Fall, 2000
x42620; dsmith@ucsd.edu

Two Examples of

Restriction Fragment Length Polymorphism

(RFLP) Analyses

Example 1 - Standard RFLP Analysis via Southern Gels and Radioactivity Assay
Example 2 - RFLP Analysis with Three Alleles, with Pedigree Analysis

Example 1 - Standard RFLP Analysis via Southern Gels and Radioactivity Assay

In this example, human DNA is treated with two different R.enzymes, A and B. These cut the DNA at different R.sites. The restricted DNA is analysed by Southern Gels, using as probe radioactive oligonucleotides complementary to the unique (single-copy) human DNA sequences shown in light green. The mutation, naturally occuring in the population, occurs as a point mutation (single nucleotide mutation) within a R.site for enzyme A, namely, site a2. This mutation is thus a SNP (Single Nucleotide Polymorphism) that gives rise to an RFLP (Restriction Fragment Length Polymorphism). If this is DNA from an autosomal chromosome, a given human will either be homozygous or homozygous for this mutation. If heterozygous, the a2 allele that permits cutting with enzyme A will be present on one chromosome, and the a2 allele that is resistant to enzyme A will be present on the other sister chromosome; when DNA from this person is cut with enzyme A and analysed via this Southern, the two Enzyme A bands shown above, corresponding to the R.fragment lengths a1-a2 and a1-a3 will be seen; this is a Polymorphism in Length of the R.fragments, i.e. an RFLP. If a person is homozygous, both chromosomes will have one or the other allele at R.site a2 and a single band will be seen in the Southern analysis of Enzyme A cut DNA, either the length a1-a2 or the length a1-a3.
Analysis with enzyme B will yield a single R.fragment length, length b1-b2, from all chromosomes from all individuals; this type of analysis is often used as a control for isolated DNA.

This is standard Southern analysis of RFLPs, and is similar to that shown in Brown, Figure 2.4

Example 2 - Standard RFLP Analysis via Southern Gels and Radioactivity Assay

In this example, RFLP analysis of the DNA from eight children, their parents, and grandparents detected the presence of three RFLP alleles for a region known to be present on chromosome 5. The DNA samples were cut with the restriction enzyme TaqI and analyzed by the Southern gel procedure. In this family, this region exists in three allelic forms characterized by TaqI sites spaced 10, 7.7, or 6.5 kb apart. Each individual has two alleles; some contain allele 2 (7.7 kb) on both chromosomes (homozygous for allele 2), and others are heterozygous at this site. Circles indicate females; squares indicate males.

a. Southern gel hybridization. Results are shown for all individuals in following columns: maternal grandmother; maternal grandfather, mother, eight children left to right, father, paternal grandmother, paternal grandfather. Note the higher intensity of the radioactivity bands for individuals that are homozygous for allele 2.

b. Possible chomosome sites and R.site mutations: R.sites and position of the Southern probe (blue rectangle) that could account for these data. Individuals with allele 1 lack TaqI sites 2 and 3; individuals with allele 2 lack TaqI site 2; and individuals with allele 3 have all TaqI sites.

c. Pedigree of the Three Generations for these Alleles. Note the Heterozygosity for these 14 individuals: 5 of the 14 are homozygous, 9 of the 14 are heterozygous. The heterozygosity for this marker and these individuals is thus 9 / 14 = 0.64; this is usefully high and informative.

[After H. Donis-Keller et al., 1987, Cell 51: 319]

If you have problems or comments, send email to Doug Smith

M W F; 10:10 - 11:00 pm	Molecular Biology	Douglas W. Smith
York 2722	BIMM 100	5254 Muir Biology Building
Fall, 2000		x42620; dsmith@ucsd.edu