Sequencing genes and genomes

Question 1

What are the two approaches to DNA sequencing?

Answer 1

Chain termination and chemical degradation - chemical degradation is not used much anymore as there are automated methods that are based on chain termination.

Question 2

What forms the basis of chain termination?

Answer 2

During the synthesis of the DNA strand the formation of the phosphodiester bond between the growing strand and the incoming nucleotide requires a 3’ hydroxyl group as well as the 5’ tri-phopshate. Without the 3’ hydroxyl, the bond cannot form.

Question 3

What do dideoxynucleotides lack?

Answer 3

The 3’ hydroxyl group.

Question 4

What happens if dideoxynucleotides are incorporated into the chain?

Answer 4

The ddNTP cannot form another phosphodiester bond and the chain terminates.

Question 5

What happens in chain termination sequencing?

Answer 5

DNA polymerase (Klenow or more specialised enzymes) is mized with the single-stranded template, primers, a supply of dNTPs and a small amount of ddNTPs (of all different bases). The result is four families of molecules that are stopped at a particular ddNTP.

Question 6

How can the sequence be read?

Answer 6

Polyacrylamide gel electrophoresis can separate the single stranded DNA molcules. If in a capillary system, it is possible to resolve ssDNA of all lengths up to 1500 nucleotides.

Question 7

What happens after the size separation?

Answer 7

The fragments are run past a fluorescence detector to determine if the molecule is ATC or G .The data is fed to a a computer and the sequence is available as a nucleotide letter sequence

Question 8

What is the problem if the data is represented graphically?

Answer 8

There is some noise underneath the peaks. This needs to be looked at to determine how reliable the sequence data is.

Question 9

Why would internal primers be used instead of a universal primer?

Answer 9

The maximum size you can sequence is 500-750 base pairs, but many internal primers based on the sequence can be synthesised for longer sequences and the total sequence is built up from the partial sequences.

Question 10

Why is there a size limit?

Answer 10

Resolving single stranded DNA molecules by electrophoresis gets more difficult as they are longer.

Question 11

How can errors be identified?

Answer 11

Sequence the same area several times and in both directions.

Question 12

What are the two approaches to genome sequencing?

Answer 12

Shotgun and clone contig.

Question 13

What happens in the shotgun sequencing method?

Answer 13

Random fragments are sequenced and the overlaps are identified. This information is used to build up a continuous genome sequence.

Question 14

What happens in the clone contig approach?

Answer 14

Overlapping clones are identified and a map is generated prior to the sequencing.

Question 15

What is a potential problem with shotgun sequencing?

Answer 15

It is random and the building up of the fragments is based only on computer programming, and is hard to tell if it’s 100% correct.

Question 16

What are other problems associated with shotgun sequencing?

Answer 16

It is based for smaller genomes as the computational aspects are complex and any identical repeated sequences can ruin the data.

Question 17

What is the pre-sequencing phase in the clone contig approach?

Answer 17

The fragments are cloned and organised into an ordered overlapping series of cloned DNA fragments.

Question 18

What is the clone contig method best for?

Answer 18

Larger genomes, but it is more expensive and time consuming.

Question 19

What cloning vectors are used in the clone contig method?

Answer 19

Vectors capable of taking very large fragments such as bacterial artificial chromosomes (BACs).

Question 20

What is the chromosome walking method used in BACs?

Answer 20

One clone is used as a hybridisation probe to the whole BAC library. The clones in which this clone hybridises with are then used to determine further overlapping clones to build up a clone contig. These clones are then each sequenced by the shotgun method.

Question 21

What method should be used if the genome of the species in question has already been sequenced?

Answer 21

Shotgun method as it is the easiest and cheapest method. The reference genome can help with the sequence assembly.

Question 22

What can happen after a genome has been sequenced?

Answer 22

Genes and their function can be identified. Most is done on a computer and requires expert systems based around knowledge of molecular biology coupled with computer power.

Question 23

How can genes potentially be identified?

Answer 23

A computer can search for an initiation codon (ATG) and a stop codon (TAA, TAG, TGA) in the same reading frame. These are likely to be genes.

Question 24

Why is identifying genes in eukaryotes harder?

Answer 24

Lots of the sequence is non-coding DNA, repeated sequences and the introns disrupt ORFs.

Question 25

How can genes be identified in eukaryote sequences?

Answer 25

Some good signposts include GC-rich regions usually occur at the start of genes, there may be codon bias that can help identify ORFs. Intron-exon boundaries can also be identified, upstream regulatory consensus sequences can also be recognised, along with training expert systems on common features of genes from a particular species or group of species.

Question 26

How can homology searches identify genes?

Answer 26

Looking at similar DNA based on common evolutionary histories of species and genes. The gene can also be translated into a sequence of amino acids and compare those.

Question 27

What are orphans?

Answer 27

Genes of unknown function.

Question 28

How can orphan genes be identified?

Answer 28

Using reverse genetics and making gene knockouts to see if there is a phenotypic change that can provide a clue of their function. This is easier in some species, such as yeast, compared to others.