Note the questions that need answering are listed in Bold Print. Figures 2 is th

Question

Note the questions that need answering are listed in Bold Print. Figures 2 is the table; figure 4 is the diagram at the bottom!

Sickle Cell Anemia Detection Simulation: PCR, Restriction Enzyme Digest, and Electrophoresis

In this activity, the genotyping of a newborn baby will be simulated with respect to the sickle cell mutation. Does the baby have a wild-type beta-globin gene, a mutation on one allele (carrier), or a mutation on both alleles (homozygous)? Known samples of wild-type and homozygous mutant beta-globin will be used for comparison.

3. Determine the recognition site and cleavage pattern of Ddel by referring to the restriction endonuclease chart provided in (Figure 2) below.

Remember that the “N” notation represents any of the four nucleotides.

4. In your written DNA sequences, draw a circle around the Ddel recognition site(s) and draw a bold line where DdeI would cleave. Remember, DdeI will only cut where there is an exact match in sequence.

5. After the restriction endonuclease treatment, visualize the bands on a gel. On the gel diagram (Figure 4), indicate where the DNA fragment(s) in the wild-type and homozygous mutant beta-globin samples would be expected to run. Draw a line for each band. Use the DNA marker with known base pair sizes to orient the bands. The sample DNA fragment from the baby is drawn.

FIGURE 4

Explanation / Answer

please provide the DNA sequence of the following-

wild type beta globin gene

mutant beta globin DNA sequence.

though probably wild type will contain the RE site for Dde1 and mutant will not contain that RE site. so in wild type we will get two bands while in mutant we will get one band.

Wild-Type: 5’ CTG ACT CC/T GAG 3’

  3’ GAC TGA GGA CT/C 5’ and

Homozygous Mutant: 5’ CTG ACT CCT GTG 3’

3’ GAC TGA GGA CAC 5’

REsite for Ddn1 is C/TNAG .

in wild type we will get bands of 11bp, and 4 bp and in homozygous mutant Dde1 will not cut so we will get 12 bp band only.

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