S12) Advanced Molecular Techniques Flashcards

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1
Q

Identify the three different ways in which DNA can be analysed

A
  • At nucleotide level
  • At gene level
  • At the chromosome level
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2
Q

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

A
  • DNA sequencing
  • PCR (+ restriction analysis)
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3
Q

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

A
  • Southern hybridisation
  • Northern hybridisation
  • RT-PCR
  • Microarray
  • DNA fingerprinting/ DNA profiling
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4
Q

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

A
  • Karyotyping
  • FISH/Chromosome painting
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5
Q

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

A
  • 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?
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6
Q

What is the reverse transcription polymerase chain reaction?

A

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

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7
Q

Traditional PCR is used to exponentially amplify target DNA sequences.

How does RT-PCR differ from this?

A
  • 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
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8
Q

What is DNA profiling?

A

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

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9
Q

DNA profiling uses short tandem repeats.

What are these and what is their benefit?

A
  • 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
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10
Q

Identify 3 circumstances where DNA profiling is used

A
  • Paternity testing
  • Immigration disputes
  • Criminal investigations
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11
Q

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

A

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

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12
Q

What is Fluorescence in situ hybridization?

A

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

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13
Q

What is DNA hybridisation?

A

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

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14
Q

In 5 steps, describe the process of DNA hybridisation

A

⇒ 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

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15
Q

Identify and describe the two different types of hybridisation techniques

A
  • Southern blotting uses DNA probes to identify complementary DNA sequences after gel electrophoresis
  • Northern blotting uses DNA to detect RNA sequences after gel electrophoresis
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16
Q

In 5 steps, outline the process of Southern blotting

A

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

17
Q

Why do we use Southern hybridisation?

A
  • 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
18
Q

What are the characteristics of DNA probes in blotting?

A
  • 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
19
Q

Western blotting is not a DNA hybridisation technique.

What is it?

A

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