Sequencing Genes And Genomes

Question 1

Define DNA sequencing

Answer 1

The process of determining the precise order of nucleotides within a DNA molecule

Question 2

What is the chain termination method?

Answer 2

One of the first methods to determine the order of bases/ nucleotides in a section of DNA (gene)

Question 3

Step 1 of chain termination method

Answer 3

A mixture of the following is added to four separate tubes:
- a single stranded DNA template
- DNA polymerase
- lots of DNA primers- short pieces of DNA
- free nucleotides
- a fluorescently labelled modified nucleotide (once its added to a DNA strand no more bases are added after it
A different modified nucleotide is added to each tube (A, T, C, G)

Question 4

Step 2 of chain termination method

Answer 4

The tubes undergo PCR, producing many strands of DNA

The strands are different lengths because each one terminated at a different point depending on where the modified nucleotide was added (after modified nucleotide is added to the DNA the strand is terminated as no more bases can be added)

Question 5

step 3 of chain termination method

Answer 5

The DNA fragments in each tube are separated by electrophoresis and visualised under UV light (because of fluorescent label)

So by reading the bands from the bottom of gel upwards, you can build up the DNA sequestered one base at a time

Question 6

Disadvantage of chain termination method

Answer 6

Can only be used for DNA fragments up to about 750 bp long

Question 7

Whole Genome sequencing

Answer 7

Used if you want to sequence the entire genome (all the DNA) of an organism
- you need to chop it up into smaller pieces first
The smaller pieces are sequenced and then put back in order to give the sequence of the whole genome

Question 8

Step 1 of genome sequencing

Answer 8

A genome is cut into smaller fragments (about 100 000 by) using restriction enzymes

Question 9

Step 2

Answer 9

The fragments are inserted into bacterial artificial chromosomes (BACs) - these are man-made plasmids
Each fragment is inserted into a different BAC

Question 10

Step 3

Answer 10

The BACs are then inserted into bacteria - each bacteria contains a BAC with a different DNA fragment

Question 11

Step 4

Answer 11

The bacteria divide, creating colonies of clones cells that all contain a specific DNA fragment
Together the different colonies make a complete genomic DNA library

Question 12

Step 5

Answer 12

DNA is extracted from each colony and cut up using restriction enzymes producing overlapping pieces of DNA

Question 13

Step 6

Answer 13

Each piece of DNA is sequenced, using the chain termination method

Question 14

step 7

Answer 14

The pieces are put back in order to give the full sequence fro that BAC

Question 15

Step 8

Answer 15

Finally the DNA fragments from all the BACs are put back in order by computers to complete the entire genome

Question 16

Sequencing and synthetic biology

Answer 16

By sequencing a gene, the sequences of amino acids that a gene codes for and so the primary structure of a polypeptide can be predicted (because 3 DNA bases codes for an amino acids)

This has allowed us to create biological molecules from scratch - synthetic biology

Question 17

What is computational biology?

Answer 17

Using computers to study biology
Allows us to compare genome sequences between organisms of different species and same species

Question 18

What is bioinformatics?

Answer 18

Developing and using computer software that can analyse, organise, and store biological data

Question 19

What can comparison of gene sequences and genomes be used for?

Answer 19

• predicting an organism’s phenotype by analysing its genotype
• epidemiological studies (the study of health and disease within a population, some gene mutations have been linked to greater risk of disease) computerised comparison between genomes of people that have a disease and those that don’t can be used to detect particular mutations that could be responsible for increase risk of disease
• understanding evolutionary relationships (tell us how closely related different species are, if the evolved from a common ancestor)