DNA Sequencing

Question 1

Dideoxyribonucleotide triphosphates (ddNTPs)
cause chain termination during DNA synthesis

Answer 1

Synthesising a DNA strand in vitro requires:
* a primer that base pairs with the DNA template to be sequenced
* DNA polymerase to extends the primer, synthesises a new DNA strand
* dNTPs as building blocks
* ddNTPs can be incorporated into DNA
- ddNTPs cause chain termination due to the lack of a 3’ OH on the sugar

Question 2

Dideoxyribonucleotide triphosphates (ddNTPs)
cause chain termination during DNA synthesis photo

Answer 2

Question 3

Modern sequencing is based on Sanger DNA sequencing but is done in one reaction tube using fluorescently labelled ddNTPs

Answer 3

• The four ddNTPs are labelled with four different fluorescent labels
• The DNA synthesis is done in the presence of all four dNTPs and all four labelled ddNTPs in one tube
• ddNTPs cause chain termination
• The synthesised products are separated by size using capillary gel electrophoresis →
  shortest products run fastest
• A laser excites the fluorescent labels.
• The fluorescent labels emit a label specific light that is detected
• A computer records the label specific signal over time and produces a chromatogram

Question 3

Sanger sequencing of DNA makes use of ddNTP chain termination

Answer 3

Question 3

Identifying DNA fragments in simple DNA samples/genomes photo

Answer 3

Question 4

Reading a sequence chromatogram photo

Answer 4

Question 5

Restriction Enzyme Mapping photo

Answer 5

Question 6

Identifying DNA fragments in complex DNA samples/genomes

Answer 6

Digestion of complex genomes with restriction enzymes creates thousands of fragments differing by size
The smear visualised in the agarose gel of digested complex
DNA is due to DNA fragments with small size differences migrating very closely together
How can we identify a specific DNA fragment in the comolex mixture?
By hybridising a labelled probe to the DNA fragment
Hybridisation is used to detect specific DNA fragments in
UNA dels or specific KNAS In KNA gels

Question 7

Identifying DNA fragments in complex DNA samples/genomes photo

Answer 7

Question 8

Hybridization: forming a hybrid of two nucleic acid molecules

Answer 8

Hybrid of a probe to a DNA strand
Probes
Single stranded. usualv DNA
15 to 1000’s of nucleotides long
Hybridize to single stranded DNA molecules
Hybridization temperature impacts on outcome
> Homologous probes: to detect identical nucleic acid molecules, 100% complementation at higher temperatures
* Heterologous probes: to detect related nucleic acid molecules. lower temberature used
> Formamide lowers melting temperature of
nucleic acid dublexes - laciitates the tormation of complementary strands

Question 8

Nuclei Acid Blotting - Separating Nucleic Acids photo

Answer 8

Question 8

Hybridisation is based
on denaturation and renaturation of DNA photo

Answer 8

Question 9

Hybridization: forming a hybrid of two nucleic acid molecules photo

Answer 9

Question 9

Hybridisation techniques

Answer 9

• Hybridisation techniques are used to detect specific DNA and RNA molecules
  Examples
• For DNA:
• Southern blotting (Edwin Southern) - gel based, uses a blot and hybridisation
• Fluorescent in situ hybridization (FISH) - cellular based, hybridisation done using tissue sections
• For RNA:
• northern blotting - gel based, uses a blot and hybridisation
• In situ hybridisation - cellular based, hybridisation done using tissue sections

Question 9

Nuclei Acid Blotting - Separating Nucleic Acids

Answer 9

For DNA:
calate iNA trom collc
Digest with a restriction enzyme
pedalae usine V.o% - 4.070
agarose gel
migrates as double stranded (ds)
SONA
13.600 be
For RNA
Isolate RNA from cells
separate using %-1.9% agarose
gel in the presence of a denaturing agent
Removes secondary structures from RNA
Ensures that RNA migrates as single stranded molecule

Question 9

Southern and northern Blotting - overview photo

Question 10

Southern and northern blotting
- hybridising a labelled probe to the DNA or RNA of interest photo

Answer 10

Question 11

Autoradiogram of a Southern blot with radioactive labelled probe photo

Answer 11

Question 12

Autoradiogram of a northern blot with radioactive labelled probe photo

Answer 12

Question 13

Making a probe: Labelling DNA Molecules - Random Priming

Answer 13

DNA is denatured to generate single stranded DNA that can act as templates for DNA synthesis
Hexanucleotides:
* DNA primer with six nucleotides
* Using a mixture of many variations of hexanucleotides increases the chance that any DNA molecule can be labelled
DNA polymerase adds nucleotides to the primer to synthesise complementary DNA strands.
As some of the nucleotides used were labelled, it results in a population of DNA molecules that contain labelled examples of all sequences of both DNA strands

Question 14

Making a probe: Labelling DNA Molecules - Random Priming photo

Answer 14

Question 15

Making a probe: Molecules used to label DNA probes photo

Answer 15

Question 16

Detection of Chemically Labelled Hybridisation Products photo

Answer 16

Question 17

Fluorescent in situ hybridisation (FISH) photo

Answer 17

Question 18

In situ Hybridisation for RNA detection - using antisense probes photo

Answer 18

Question 19

Can you …

Answer 19

• explain how Sanger sequencing works and why it uses ddNTPs?
• relate Sanger DNA sequencing to DNA replication?
• read a DNA sequence and the reverse complement sequence from a sequencing gel and a chromatogram?
• can you explain the principal idea of a restriction digest map of DNA?
• explain what happens to double stranded DNA (or RNA) if you denature or renature it?
• explain conditions for the hybridisation of a homologues and a non-homologous probe to DNA or RNA?
• compare Southern and northern blot hybridisation, what they detect and how they work?
• recognise and read a Southern and northern blot?
• explain different methods and labels used for the detection of nuclei acids in Southern and northern blots?
• outline experimental steps and probes used in in situ hybridisation and in Fluorescent in situ hybridisation to detect DNA or RNA?