18 - Advanced Molecular Techniques

Question 1

How does DNA hybridisation occur?

Answer 1

• Heat up DNA to form ssDNA by denaturation
• Add a complemntary ssDNA probe with a radioactive/fluorescent marker and cool the mixture
• Probe will anneal to the DNA and hybridise, can identify DNA on photographic film

Question 2

What are Northern, Western, Southern blotting identifying?

Answer 2

Western is not a DNA hybridisation technique like the others

Question 3

What is the methodology for a southern blot?

Answer 3

Question 4

What are the characteristics of a probe in a Southern blot?

Answer 4

Question 5

What is the methodology of a Nothern blot?

Answer 5

Same as Southern but with RNA and DNA detects the RNA

Question 6

What is DNA sequencing?

Answer 6

• Working out the genome using the Sanger chain termination method
• ddNTP blocks elongation as no further phosphodiester bonds can be formed

Question 7

What happens to restriction sites in sickle cell?

Answer 7

The mutation causes the 6th amino acid to be a misfunctional restriction site so can use DNA gel electrophoresis to show different length band

Question 8

What are some ethical issues with DNA sequencing?

Answer 8

• Who owns DNA?
• Discrimination? (e.g employers and insurance companies, spouse, police)
• Can knowledge help prevent illness or treatments)

Question 9

What are allele specific primers?

Answer 9

Question 10

What is RT-PCR?

Answer 10

Question 11

What is DNA microarray?

Answer 11

• 1000’s of genes and can compare two conditions
• Fluorescently labelled DNA

Question 12

What are the two types of microarray technology?

Answer 12

Question 13

How is DNA Fingerprinting/Profiling done?

Answer 13

• Look at microsatellites (non-coding tandem repeats) as they have high variability in each person
• Non-coding repeats are inherited so will be similar to parents
• Likelihood of same person having the same tandem repeats at each microsatellite very small

Question 14

What is karyotyping?

Answer 14

Looking at chromosome level

• Look at chromosomes in metaphase spread and their banding patterns and pair up

Question 15

What is FISH?

Answer 15

• Denature and hybridise DNA with fluorescent probe, look for microdeletions at chromosome level that are too small to see karyotyping
• Use a control probe
• Just inject probe into cell

Question 16

What is chromsome painting?

Answer 16

• Family of probes each with same fluorescent wavelength tag if gene on same chromosome
• Can see transolcations

Question 17

What are the ethical issues behind embryo genetic testing?

Answer 17

• May terminate pregnancy based on disease, lowers value of life
• May give information about people who are already alive and they may not want to know, e.g Huntington’s Disease