DNA Hybridisation

Question 1

Explain how a DNA probe can be added to DNA

Answer 1

• dsDNA is denatured by heat and H bonds break
• ssDNA added which is complementary to one of the strands and contains a fluorescent marker
• When ssDNA molecules reanneal, some do so with the marked ssDNA so can be identified using photographic film

Question 2

Outline the main steps in Southern blotting

Answer 2

• DNA fragments separated by electrophoresis
• Fragments transferred onto a nitrocellulose membrane
• hybridisation of a probe to detect a specific piece of DNA
• Visualisation of marked DNA probe using photographic/X-ray film

Question 3

How is DNA denatured in Southern blotting?

Answer 3

DNA fragments in gel are soaked in alkali solution, breaking H bonds between complementary bases

Question 4

State 2 ways in which the DNA in gel electrophoresis can be transferred onto a filter

Answer 4

• Capillary action for the gel onto filter

- Electrophoretic transfer -> apply voltage across set-up so -ve DNA moves from gel to filter

Question 5

Why is the DNA on the nitrocellulose filter single stranded?

Answer 5

Before the DNA fragments are transferred onto the filter, the gel is soaked in alkali solution which causes the dsDNA to denature into ssDNA

Question 6

Describe the action of an oligonucleotide synthesier

Answer 6

Produces DNA probes from free dNTPs using a programmed sequence

Question 7

Why do we use Southern blotting?

Answer 7

• Allows us to detect pieces of DNA from complex mixtures that may be difficult to detect
• Allows us to detect very small pieces of DNA which may not be visible by staining

Question 8

Why is Southern blotting useful at the gene level?

Answer 8

• Investigate gene structure e.g. large deletions or duplications
• Investigate gene expansions or triplet repeats
• Investigate mutations in genetic tests using allele specific probes e.g. Sickle cell disease
• Investigate variation and genetic relationships

Question 9

Give 3 characteristics of DNA probes in blotting

Answer 9

• Probes are not 100% complementary to target sequence (bind, but less tightly)
• Probes do not have to completely align with target sequence
• Probes do not affect position of target sequence on gel

Question 10

What is the purpose of the Sanger Chain Termination method?

Answer 10

• DNA sequencing

- Identify the nucleotide sequence of a piece of DNA

Question 11

How does a deoxynucleotide (dNTP) differ from a dideoxynucleotide (ddNTP)?

Answer 11

dNTP has an OH group on C3 which can form phosphodiester bonds with adjacent deoxyribose sugars however this OH group is not present on ddNTP

Question 12

Describe the action of ddNTP

Answer 12

• Can be used by DNA polymerase as a substrate
• If incorporated it can block DNA elongation as the absence of the OH group means phosphodiester bonds cannot form with subsequent dNTPs

Question 13

Briefly describe how the Sanger Chain Termination method is used to determine the nucleotide sequence of DNA

Answer 13

• 4 separate tubes laid out each containing a modified ddNTP (A,C,T,G)
• Depending on where ddNTP is incorporated in the DNA will determine the size of the fragment
• Fragments separated on gel by basis of size (smallest at bottom)
• Fragments enter tubes which correspond to ddNTP at end of chain (e.g. If ddNTP added was ddATP then fragment would enter the A tube)
• Fragments vary in size and eventually the entire base sequence can be mapped

Question 14

Describe the process of DNA hybridisation

Answer 14

• dsDNA is heated and denatured (H bonds are broken, forming 2 ssDNA molecules)
• Mixture is then cooled and the ssDNA molecules can reanneal forming dsDNA
• 2 strands of ssDNA are complementary