18. Advanced molecular techniques

Question 1

Give the 2 ways to analyse DNA at a nucleotide level

Answer 1

1. DNA sequencing

2. PCR plus restriction analysis

Question 2

Give the 5 ways to analyse DNA at a gene level

Answer 2

1. Southern hybridisation
2. Northern hybridisation
3. Reverse transcriptase-PCR
4. Microarray
5. DNA fingerprinting/DNA profiling

Question 3

Give the 2 ways to analyse DNA at a chromosome level

Answer 3

1. Karyotyping

2. FISH / Chromosome painting

Question 4

Describe DNA sequencing

Answer 4

DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.
It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long “consensus.”

What is genome sequencing?

Question 5

What is genome sequencing?

Answer 5

This is a technique used to determine the nucleotide base sequence of the entire genome of an organism

Question 6

Give the ethical issues associated with DNA and genome sequencing

Answer 6

There are questions surrounding privacy and access of information born out of genome sequencing. For example who owns the DNA sequence information produced the individual or the sequencing company.

There are also questions on whether this information can open up areas for discrimination for example by insurance companies.

Question 7

What is polymerase chain reaction (PCR)

Answer 7

A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides.

Question 8

What is Allele specific PCR?

Answer 8

Allele specific PCR is a technique that can be used to detect single nucleotide changes this is done by designing primers which move to the region containing the mutation/polymorphism.

Question 9

What is southern hybridisation?

Answer 9

A Southern blot is an analysis method used in molecular biology for the detection of a specific DNA sequence in DNA samples.

Question 10

describe southern hybridisation

Answer 10

• digest DNA with restriction enzyme
• seperate DNA fragments by gel electrophoresis
• gel soaked in alkaline solution to denature DNA to ssDNA molecules
• transfer ssDNA fragments to nylon or nitrocellulose filter membrane
• then the subsequent fragment detection by probe hybridisation (a single DNA fragment with a target-allele specific sequence whose presence in the target DNA is being determined).
• wash filter to remove any unbound probe
• The pattern of hybridisation is visualised on X-ray film by autoradiography (in the case of a radioactive or fluorescent probe),
• or by development of colour on the membrane (if a fluorescence detection method is used).

Question 11

why use southern hybridisation?

Answer 11

• to investigate gene structure
• to investigate gene expansions, triplet repeats
• to investigate mutations in genetic tests
• to investigate variation, genetic relationships