48. A schematic representation of restriction fragment length polymorphism (RFLP) analysis of a sample population is shown below. The number of people exhibiting a given pattern is indicated above the lanes.

Calculate the frequency of 6.5 kb allele. [Correct to two decimal places]

RFLP Analysis: Calculate the Frequency of the 6.5 kb Allele

Correct Answer

Answer: 0.21

The frequency of the 6.5 kb allele is calculated by counting the total number of copies of the 6.5 kb allele in the population and dividing this value by the total number of alleles present in the population. The RFLP diagram contains six different banding patterns representing groups of 8, 40, 20, 7, 13, and 12 people.

The total population size is:

8 + 40 + 20 + 7 + 13 + 12 = 100 people

Since every person is diploid and therefore carries two alleles at the locus being analyzed, the total number of alleles in the population is:

2 × 100 = 200 alleles

The 6.5 kb band is present in three groups containing 8, 20, and 13 people. Each of these individuals is heterozygous and therefore contributes one copy of the 6.5 kb allele.

Thus, the total number of 6.5 kb alleles is:

8 + 20 + 13 = 41 copies

The allele frequency is therefore:

Frequency of 6.5 kb allele = 41 / 200 = 0.205

Correct to two decimal places:

Frequency of 6.5 kb allele = 0.21

Understanding the RFLP Pattern Given in the Question

Restriction Fragment Length Polymorphism, commonly abbreviated as RFLP, is a molecular genetic technique based on differences in the lengths of DNA fragments generated by restriction enzyme digestion. Variations in DNA sequences can create or eliminate restriction sites or alter the distance between restriction sites, producing DNA fragments of different lengths.

In the given population analysis, each horizontal band represents a restriction fragment of a particular size. The fragment sizes shown are 6.5 kb, 5.0 kb, 4.0 kb, and 3.0 kb. Because each individual is diploid, an individual can carry two copies of the same allele or two different alleles.

An individual with two identical alleles shows a single band and is homozygous. An individual with two different alleles shows two bands and is heterozygous. Therefore, the RFLP pattern can be used to identify the genotype represented by each lane.

Reading Each Lane of the RFLP Diagram

First Pattern: 8 People

The first lane contains two bands, one at 6.5 kb and another at 3.0 kb. Therefore, these 8 people are heterozygous and have the genotype:

6.5 kb / 3.0 kb

Each person carries one copy of the 6.5 kb allele. Therefore, this group contributes:

8 × 1 = 8 copies of the 6.5 kb allele

Second Pattern: 40 People

The second lane contains two bands, one at 5.0 kb and another at 4.0 kb. Therefore, these 40 people have the genotype:

5.0 kb / 4.0 kb

The 6.5 kb band is absent from this pattern. Therefore, these 40 people contribute:

0 copies of the 6.5 kb allele

Third Pattern: 20 People

The third lane contains bands at 6.5 kb and 5.0 kb. Therefore, these 20 people are heterozygous and have the genotype:

6.5 kb / 5.0 kb

Each person carries one copy of the 6.5 kb allele. Therefore, this group contributes:

20 × 1 = 20 copies of the 6.5 kb allele

Fourth Pattern: 7 People

The fourth lane shows only one band at 4.0 kb. Since RFLP is treated as a codominant marker in this question, a single band indicates that both alleles have the same fragment length. Therefore, these individuals are homozygous:

4.0 kb / 4.0 kb

The 6.5 kb allele is absent. Therefore, this group contributes:

0 copies of the 6.5 kb allele

Fifth Pattern: 13 People

The fifth lane contains two bands, one at 6.5 kb and another at 4.0 kb. Therefore, these 13 people have the genotype:

6.5 kb / 4.0 kb

Each individual contributes one copy of the 6.5 kb allele. Therefore, this group contributes:

13 × 1 = 13 copies of the 6.5 kb allele

Sixth Pattern: 12 People

The final lane contains two bands, one at 5.0 kb and another at 3.0 kb. Therefore, these 12 people have the genotype:

5.0 kb / 3.0 kb

The 6.5 kb allele is absent from this genotype. Therefore, these individuals contribute:

0 copies of the 6.5 kb allele

Complete Genotype Interpretation of the Population

The first group contains 8 individuals with the 6.5 kb/3.0 kb genotype. The second group contains 40 individuals with the 5.0 kb/4.0 kb genotype. The third group contains 20 individuals with the 6.5 kb/5.0 kb genotype. The fourth group contains 7 individuals with the 4.0 kb/4.0 kb genotype. The fifth group contains 13 individuals with the 6.5 kb/4.0 kb genotype, while the sixth group contains 12 individuals with the 5.0 kb/3.0 kb genotype.

Only three groups contain the 6.5 kb allele:

8 people with 6.5 kb/3.0 kb

20 people with 6.5 kb/5.0 kb

13 people with 6.5 kb/4.0 kb

Therefore, the number of copies of the 6.5 kb allele is:

8 + 20 + 13 = 41

Step-by-Step Calculation of the 6.5 kb Allele Frequency

Step 1: Calculate the Total Number of People

The numbers written above the six RFLP patterns are 8, 40, 20, 7, 13, and 12.

Therefore:

Total number of people = 8 + 40 + 20 + 7 + 13 + 12

Total number of people = 100

Step 2: Calculate the Total Number of Alleles

Humans are diploid organisms, which means that each individual carries two alleles at an autosomal locus. Therefore, 100 individuals contain:

Total number of alleles = 2 × 100

Total number of alleles = 200

Step 3: Count the Copies of the 6.5 kb Allele

The 6.5 kb allele is present in the groups containing 8, 20, and 13 people.

All three patterns are heterozygous because each lane contains the 6.5 kb band together with another band. Therefore, each individual contributes only one copy of the 6.5 kb allele.

Number of 6.5 kb alleles = 8 + 20 + 13

Number of 6.5 kb alleles = 41

Step 4: Apply the Allele Frequency Formula

The general formula for calculating allele frequency is:

Allele frequency = Number of copies of the allele / Total number of alleles in the population

Substituting the values:

Frequency of 6.5 kb allele = 41 / 200

Frequency of 6.5 kb allele = 0.205

Step 5: Round the Answer to Two Decimal Places

The question specifically asks for the answer correct to two decimal places. Therefore:

0.205 ≈ 0.21

Hence, the frequency of the 6.5 kb allele is:

0.21

Why the Answer Is Not 0.41

The 6.5 kb allele is observed in 41 individuals out of a population of 100. Dividing 41 by 100 gives 0.41, but this value represents the proportion of individuals carrying the 6.5 kb allele, not the frequency of the allele itself.

Allele frequency must be calculated using the total number of allele copies in the population. Since every diploid individual carries two alleles, 100 individuals contain 200 alleles.

Therefore, the correct calculation is:

41 / 200 = 0.205 ≈ 0.21

This distinction between the proportion of allele carriers and the actual allele frequency is essential in population genetics.

Why Each 6.5 kb Band Contributes Only One Allele in This Question

Every lane containing the 6.5 kb band also contains a second band of a different size. The three relevant patterns are 6.5 kb/3.0 kb, 6.5 kb/5.0 kb, and 6.5 kb/4.0 kb.

These are heterozygous genotypes. Therefore, each individual in these groups has only one copy of the 6.5 kb allele.

If a lane had shown only a 6.5 kb band, the individual would be interpreted as homozygous 6.5 kb/6.5 kb and each such person would contribute two copies of the 6.5 kb allele. However, no such homozygous 6.5 kb pattern is shown in the given population.

Alternative Allele Frequency Formula

Allele frequency can also be calculated using the standard population genetics formula:

Frequency of allele A = [2N(AA) + N(Aa)] / 2N

In this question, there are no individuals homozygous for the 6.5 kb allele. Therefore:

N(6.5/6.5) = 0

The total number of heterozygous individuals carrying the 6.5 kb allele is:

8 + 20 + 13 = 41

Thus:

Frequency = [(2 × 0) + 41] / (2 × 100)

Frequency = 41 / 200 = 0.205 ≈ 0.21

Understanding RFLP as a Codominant Molecular Marker

RFLP markers are highly useful in genetic analysis because different alleles can often be identified directly from their fragment sizes. When two different alleles are present in a heterozygous individual, both fragment sizes are visible. This makes the marker codominant.

For example, an individual with a 6.5 kb allele and a 3.0 kb allele shows both bands. Similarly, an individual carrying 6.5 kb and 5.0 kb alleles shows two corresponding bands.

Because both alleles of a heterozygote can be observed, the genotype of each individual can be determined directly from the RFLP pattern. This allows accurate calculation of allele frequencies in a population.

Difference Between Genotype Frequency and Allele Frequency

Genotype frequency describes the proportion of individuals carrying a particular combination of two alleles. Allele frequency, in contrast, describes the proportion of all allele copies represented by one particular allele.

For example, the 6.5 kb/3.0 kb genotype occurs in 8 out of 100 individuals, giving a genotype frequency of 0.08. However, the frequency of the 6.5 kb allele must be calculated by counting all copies of the allele across every genotype in the population and dividing by the total number of allele copies.

This is why allele frequency calculations use a denominator of 2N for a diploid population.

Why This Question Is Important for Life Science and Biotechnology Exams

This question combines molecular biology with population genetics. Students must first interpret an RFLP gel correctly, identify the genotype represented by each banding pattern, count the number of people carrying the allele of interest, and finally apply the correct allele frequency formula.

Similar questions are frequently important for IIT JAM Biotechnology, CSIR NET Life Science, DBT JRF, GATE Biotechnology, CUET PG, and other life science examinations. The same principle can also be applied to microsatellites, allozymes, SNP genotypes, and other codominant genetic markers.

Concept Summary

The RFLP population contains 100 individuals, so the total number of alleles is 200. The 6.5 kb allele is present in three heterozygous groups containing 8, 20, and 13 people. Therefore, the total number of copies of the 6.5 kb allele is 41.

The allele frequency is calculated as:

41 / 200 = 0.205

When rounded to two decimal places:

0.205 ≈ 0.21

Final Answer

Total population = 100 people

Total number of alleles = 200

Number of copies of the 6.5 kb allele = 8 + 20 + 13 = 41

Frequency of 6.5 kb allele = 41 / 200 = 0.205

Correct to two decimal places = 0.21

Final Answer: 0.21