1 answer

Question 2: a) You determine that xpd is expressed preferentially in muscle. Your colleague wants to...

Question:

Kpnl Xbal (0) 150 Xbal 1100 Kpnl Xbal 2000 2100 Xbal 2700 Kpnl 3350 Kpnl 3850 wild type allele (xpd+) 1300 17002400 HA 4001 pAlthough only a fragment of a much larger chromosome is shown, for the purposes of this exercise, ignore the DNA flanking the

Question 2:

a)

You determine that

xpd

is expressed preferentially in muscle. Your colleague wants to know

if any of the mutations affect the stability of the RNA transcript, thereby leading to higher

turnover and lower overall mRNA levels. What techniques could you use to test her

hypothesis? List the pros and cons of each method. (hint: try to think of at least three methods

you could use)

b) You find that your collegue is correct, and that the xpda allele does have decreased mRNA levels, but xpdb and xpdc alleles appear to be transcribed at normal levels. Which of the following diagrams represent the blot you performed that led you to that conclusion.

Declan Aditi Javier Heba Declan Aditi Javier Heba C. Declan Aditi Javier Heba Declan Aditi Javier Heba 3 kb 2.1 kb 2.1 kb 2.1c)

You perform a western blot, on samples from your four tissue cultures, and find the following

results. Hypothesize the possible effects of each mutation on the final protein.

Declan Aditi Javier Heba 100 KD - 75 KD 50 kD 30 kD 25 kD

Kpnl Xbal (0) 150 Xbal 1100 Kpnl Xbal 2000 2100 Xbal 2700 Kpnl 3350 Kpnl 3850 wild type allele (xpd+) 1300 17002400 HA 4001 point mutation in allele xpda # V L 3200 1 3700 point mutation in allele xpdc 0.4 kb region deleted in allele xpdo
Although only a fragment of a much larger chromosome is shown, for the purposes of this exercise, ignore the DNA flanking the outer Kpnl restriction enzyme cut sites. Kpnl and Xbal restriction enzyme cut sites are shown by the vertical lines. The numbers below each line indicate the number of nucleotides from the first Kpnl site (at position 0) in the wild type allele, and the exon/intron boundarys are also listed in reference to the first cut site (so, for example, the first exon begins 400 bp after the first Kpnl cut site, and is 900 bp long (1300- regions of exons are shown by diagonal stripes, while the coding regions are solid color. You are studying Garnet-Feist syndrome, a newly identified neurodegenerative and muscle wasting disease. Using a GWAS (Genome wide association study) you have identified three mutations in the xpd gene which associate with the disease. The first, called the xpda allele, is a point mutation in the first exon (indicated by an asterisk in the image above). The second, called the xpdb allele, is a much larger change, and is caused by a deletion of 400 basepairs (0.4 kb) from exon 2. The region deleted from exon 2 is indicated by the black double bar drawn below exon 2 in the diagram above. The third, called the xpdc allele, is a point mutation in exon 3 indicated by the pound sign (and is in the middle of the Kpnl recognition sequence). You will be using several of the molecular methods covered in the textbook and lecture to study the xpd DNA, RNA and proteins of four indivuals: Declan is homozgous for the wild type allele (+/+), Aditi is heterozygous for the xpdb allele (xpdb/+), Javier is homozygous for the xpda allele (xpda/ xpda), and Heba is homozygous for the xpdc allele (xpdc/ xpdc). You have collected skin biopsies from each individual, and are using stem cell reprogramming techniques to make immortal cell culture lines to test potential therapies for GF sydrome. Unfortunately, your lab assistant labeled the cultures with a unique number identifier, but then lost the list indicating which cell culture line is derived from each person.
Declan Aditi Javier Heba Declan Aditi Javier Heba C. Declan Aditi Javier Heba Declan Aditi Javier Heba 3 kb 2.1 kb 2.1 kb 2.1 kb TI 1.7 kb 1.7 kb 1.7 kb 1.1 kb 1.1 kb 0.5 kb 0.5 kb 0.5 kb 0.5 kb
Declan Aditi Javier Heba 100 KD - 75 KD 50 kD 30 kD 25 kD

Answers

a) In order to check the effect of mutations on the stability of RNA , the following methods can be used:

1) Northern blotting:This method is used to check the shape and relative stability of an RNA in a particular complex sample.The mRNAs therefore can be analysed.

PROS:

A large number of genes can be subjected to analysis at a time.

They are rapid,comparitively cheap and less technical knowledge is required.

CONS:

A very high tendency of degradation for these molecules.

The use of multiple probes may be problematic leading to less accuracy.

2)In Situ Hybridisation: It is amethod used to accurately localize a particular nucleic acid be it DNA or RNA in a givenhistologic section.

PROS:

Libraries of the tissues can be formed.

They can be stored in for future used.

CONS:

Difficulty in identification targets with trace amounts.

3) Nuclease Protection Assays (NPA): This methods identifies a specific RNA from a sample of heterogenous RNAs

PROS:

The have shown to be more tolerant to partially degraded RNA

Multi probe assays are easy with NPA

CONS:

The transcript size may not be recorded.

Lacks probe flexibility.

b) D is the correct blot for the situation here Javier is homozygous for xpda allele and in D there depiction of decreased levels so the blot from Javier is transparent in the fourth blot and third blot.Also the xpdb allele is expressed in normal terms and as for aditi is not decresed as shown in the C option.So D is the correct blot.

c) Since a thinner band is observed in Javier's blot depicting that xpda allele has undergone mutation.

.

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