S12) Advanced Molecular Techniques

Question 1

Identify the three different ways in which DNA can be analysed

Answer 1

• At nucleotide level
• At gene level
• At the chromosome level

Question 2

Which two techniques can be used to analyse DNA at nucleotide level?

Answer 2

• DNA sequencing
• PCR (+ restriction analysis)

Question 3

Which five techniques can be used to analyse DNA at gene level?

Answer 3

• Southern hybridisation
• Northern hybridisation
• RT-PCR
• Microarray
• DNA fingerprinting/ DNA profiling

Question 4

Which two techniques can be used to analyse DNA at chromosomal level?

Answer 4

• Karyotyping
• FISH/Chromosome painting

Question 5

Identify the 4 questions which revolve around the ethics of DNA sequencing

Answer 5

• Who would be interested in your genome information?

I. Family/potential spouse

II. Doctor

III. Government

IV. Police

IV. Insurance companies

• Can the knowledge help prevent illness later in life?
• Does it open up areas for discrimination?
• Who owns DNA sequence?

Question 6

What is the reverse transcription polymerase chain reaction?

Answer 6

RT-PCR, a variant of polymerase chain reaction, is a technique commonly used to detect RNA expression by producing multiple copies of particular DNA isolates through amplification

Question 7

Traditional PCR is used to exponentially amplify target DNA sequences.

How does RT-PCR differ from this?

Answer 7

• RT-PCR clones expressed genes by reverse transcribing the target RNA into its DNA complement using a reverse transcriptase
• Thereafter, the newly synthesised cDNA is amplified using traditional PCR

Question 8

What is DNA profiling?

Answer 8

DNA profiling (aka DNA fingerprinting) is a forensic technique used to identify individuals by characteristics of their DNA

Question 9

DNA profiling uses short tandem repeats.

What are these and what is their benefit?

Answer 9

• Short tandem repeats are highly variable repetitive sequences
• The STR loci are very similar between closely related humans, but are so variable that unrelated individuals are extremely unlikely to have the same

Question 10

Identify 3 circumstances where DNA profiling is used

Answer 10

• Paternity testing
• Immigration disputes
• Criminal investigations

Question 11

In 6 steps, outline the procedure for creating a DNA fingerprint

Answer 11

⇒ DNA is extracted from sample cells e.g. skin, hair, blood and purified

⇒ DNA cut into fragments using restriction enzymes

⇒ Gel electrophoresis of dsDNA fragments (distributes according to length)

⇒ dsDNA fragments are blotted & ssDNA fragments are transferred to a nylon sheet

⇒ Autoradiography using DNA probes

⇒ Develop, visualise and analyse DNA fingerprint

Question 12

What is Fluorescence in situ hybridization?

Answer 12

FISH is a cytogenic technique that uses fluorescent probes which bind only to the parts of the chromosome with a high degree of sequence complementarity

Question 13

What is DNA hybridisation?

Answer 13

DNA–DNA hybridisation is a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences

Question 14

In 5 steps, describe the process of DNA hybridisation

Answer 14

⇒ dsDNA is denatured (heated / treated with an alkaline solution)

⇒ H₂ bonds break & ssDNA is released

⇒ Add identical ssDNA labelled with a radioactive/fluorescent marker

⇒ Anneal DNA

⇒ Identify labelled DNA using photographic film

Question 15

Identify and describe the two different types of hybridisation techniques

Answer 15

• Southern blotting uses DNA probes to identify complementary DNA sequences after gel electrophoresis
• Northern blotting uses DNA to detect RNA sequences after gel electrophoresis

Question 16

In 5 steps, outline the process of Southern blotting

Answer 16

⇒ Digest DNA with restriction enzymes

⇒ Gel electrophoresis to separate DNA fragments

⇒ Transfer to a nylon membrane

⇒ Hybridisation of a probe to detect target DNA

⇒ Use X-ray film to visualise labelled probe & target DNA

Question 17

Why do we use Southern hybridisation?

Answer 17

• To investigate gene structure e.g. large deletions/duplications
• To investigate gene expansions e.g. Huntington’s disease
• To investigate mutations in genetic tests e.g. Sickle cell disease
• To investigate variation & genetic relationships e.g. DNA fingerprinting

Question 18

What are the characteristics of DNA probes in blotting?

Answer 18

• Probes don’t have to have 100% similarity to the target sequence.
• Probes don’t have to completely align with the target sequence
• Probes don’t affect position of the target sequence on a gel

Question 19

Western blotting is not a DNA hybridisation technique.

What is it?

Answer 19

Western blotting involves the detection of proteins by antibodies after protein gel electrophoresis