L6

Question 1

SNPs can induce 4 types of mutations:

Answer 1

1. Neutral (altered codon encodes same a.a)
2. Altered codon encodes chemical similar a.a (may not significantly affect protein structure/function)
3. Altered codon encodes a chemically different a.a (likely to affect protein structure/function)
4. Altered codon is stop codon (often produces completely inactive protein)

Question 2

How would we detect the effect of a SNP on protein synthesis?

Answer 2

Wester blotting

Question 3

What is Wester blotting?

Answer 3

• Uses whole-cell/whole-tissue protein samples
  1. Run sample on polyacrylamide gel
  2. Transfer the protein on the gel onto a membrane
  3. Incubate membrane with primary antibody specific for protein of interest
  4. Incubate membrane with a fluorescently labelled secondary antibody

Question 4

INDELs in protein-coding region

Answer 4

• INDELs often lead to frameshift mutation
• Effect of frameshift mutation depends on where the INDEL occurs in the protein-coding region, and the sequence downstream of the mutation
• May result in mild-to-severe changes in structure and function

Question 5

Intron boundaries

Answer 5

• All introns must be removed before translation
• Almost all introns begin with GU and end with AG
• High conservation of intron and exon nucleotides adajcent to GU and AG
• If GC changed to GU, then part of the exon will be spliced out with intron = less a.a
• Or if GU changed to TU, the intron won’t be spliced = extra a.a

Question 6

SNPs, SSLPs, and INDELs in regulatory elements can:

Answer 6

1. Prevent the transcriptional machinery from recognizing the promoter (no transcription = no protein)
2. Increase or lession the recruitment efficiency of the transcriptional machinery to the promoter (increase or decrease transcription = increased or decreased protein level)
3. Increase or lessen the result efficiency of transcriptional factors to enhancers (increase or decrease transcription = increased or decreased protein level)

Question 7

Reverse transcriptase qPCR (RT-qPCR)

Answer 7

• Uses mRNA to directly measure the transcriptional activity of a gene
  1. Isolate mRNA from tissue or cell sample
  2. Reverse transcribe it into cDNA
  3a. Use PCR to amplify a specific cDNA molecule that represents the gene of interest and gel electrophoresis
  3b. Perform qPCR to quantify transcription of the gene of interest

Question 8

Quantitative PCR (qPCR)

Answer 8

• Allows you to measure the relative amount of a particular segment of DNA in a sample
  Differences between PCR and qPCR
  1. Addition of dye that emit fluorescence when inserted into double-stranded DNA
  2. Real-time PCR reader