DNA sequencing

Question 1

Which molecular analytical techniques rely on DNA sequencing?

Answer 1

• PCR (to make primers)
• Recombinant techniques (cloning and protein expression)
• Protein structure and function (mutagenesis)
• Species identification
• Molecular phylogeny understanding
• Evolutionary analysis
• Biotechnology (e.g., the production of polymerases)

Question 2

Who invented the process of DNA sequencing?

Answer 2

Sanger -> he also invented the process of sequencing proteins/amino acids

Question 3

What are ddNTPs and how do they work?

Answer 3

They are dideoxynucleotides, which inhibit the nucleotide chain from elongating:
ddATP, ddGTP, ddTTP, ddCTP

They lack a hydroxyl (OH) group on the 2’ and 3’ positions on the ribose sugar, which the next nucleotide would bind to via a polymerase reaction to form phosphodiester bonds. They are also therefore known as 2’, 3’ ddNTPs.

They essentially terminate the elongation of a strand, causing the DNA polymerase to drop off

Question 4

As a recap, how are nucleotides added to DNA strands?

Answer 4

The new nucleotide binds with its alpha phosphate to the OH group of the above nucleotide on the 3’. This causes the triphosphate tail of the new nucleotide to be cleaved off and an phosphodiester bond is formed

Question 5

How is the Sanger method carried out?

Answer 5

• Four different polymerase mixes are produced
• Each one contains an unknown template sequence of single stranded DNA, DNA polymerase, dNTP (deoxynucleotides of each type, e.g., dATP, dGTP, dCTP, dTTP), a radioactively labelled primer (this was only in the original method)
• These mixes are added to four different reaction mixes each containing a different type of ddNTP (ddATP in one, ddCTP etc.)
• Therefore, the chains in each different mixture will be ended with the base of the ddNTP present
• The fragments can then be run via gel electrophoresis. The shortest fragments travel the furthest through the gel, meaning you start the sequence with these

Question 6

How has the Sanger method changed? What does it produce?

Answer 6

• Fluorescently labelled ddNTPs (containing a fluorochrome) are added instead of radioactive phosphorous. This means that at each termination, a fluorescent label is present, visible under a laser.

Different colours of fluorochromes are now added for each ddNTP so the same mix and therefore only one lane can be used to run the results -> this makes it cheaper and allowed faster sequencing

This produces a primary sequence diagram (like little different coloured peaked waves). The first part of the diagram is when they are too small to properly dissolve. The peaks can be used to deduce the base and therefore the sequence.

Question 7

How does the Sanger method cover every sequence position?

Answer 7

• ddNTP amounts are calculated to statistically induce elongation stops in every sequence position

Question 8

What does it mean if two peaks are over the top of each other in a primary sequence diagram?

Answer 8

This suggests that mutations have occurred during the process (e.g., via mistakes by DNA polymerase during PCR)

Question 9

What does a Sanger diagram produce (modern technique)?

Answer 9

• Primary sequence diagram

Question 10

Summarise the steps required for the Sanger method.

Answer 10

1. PCR amplification of the region of interest
2. Chain termination PCR using fluorescently labelled ddNTPs
3. PCR fragments separated based on size in a gel and read in the order in which fluorescent signals are detected

Question 11

What other method does the Sanger method rely on?

Answer 11

It is a modification of PCR

Question 13

What is another technique that can be used for genetic sequencing? (don’t worry about detail here)

Answer 13

Second generation sequencing

Question 14

How can differences in individuals be identified? Think of case study.

Answer 14

Using Internal Transcribing Spaces (ITS).
- These are non-coding and therefore more mutations occur here as it does not affect the functioning of an organism.

1. This DNA is amplified and sequenced using sequencing chromatography
2. The resulting sequences can be used to search on GenBank (which uses a Basic Local Alignment Search Tool (BLAST)), for example, to find out the meaning of certain sequences and alignment with already known sequences

One example is the study of thermotolerant symbionts, such as the C3 strain in the PAG

Question 15

How can phylogenetic trees be generated?

Answer 15

Using Multiple Sequence Analysis?
- Non-coding regions of a certain strain can be amplified (PCR) and sequenced
- These are then put into data bases such as GenBank and compared to other strains
- Phylogenetic trees can be produced from this to show suggested evolutionary changes