MG (DNA sequencing)

Question 1

what’s DNA sequencing

Answer 1

technique that allows for the nucleotide base sequence of an organism to be identified and recorded

Question 2

what sequencing method was developed by Frederick Sanger in the 70s and what does it involve?

Answer 2

• chain termination method also known as Sanger sequencing
• involves synthesising many DNA fragments that differ in length by one base pair from a DNA template strand

Question 3

what’s needed for Sanger sequencing ?

Answer 3

• The single stranded DNA being sequenced.
• A mixture of ‘normal’ nucleotides (A, T, C, G)
• One type of terminator nucleotide ( lacks -OH on sugar so phosphodiester bond can’t form)
• primers
• DNA Polymerase.

Question 4

How was Sanger’s DNA sequencing carried out?

Answer 4

• extract DNA
• cut DNA with restriction enzymes to get varying lengths of DNA
• amplify using PCR
• a single strand of DNA used as a template for four experiments in separate dishes.
• Each dish contained a solution of the four bases - A,T,G and C plus the enzyme DNA polymerase.
• Each dish had a modified version (once incorporated into the synthesised complementary strand of DNA, no more bases could be added) of one of the DNA bases added to it. They’re labelled with a radioactive isotope.
• called terminators
• you get a variety of ‘partially completed’ DNA strands, because they have been ‘terminated’ at different points.

Question 5

How is the single strand used in chain termination method complementary to the template strand made?

Answer 5

• primer anneals to the start of the single stranded template, producing a short section of double stranded DNA at the start of the sequence
• DNA polymerase attaches to this double stranded section and begins DNA replication using the free nucleotides in the test tube
• H bonds form between the complementary bases on the nucleotides
• Once the incubation period has ended the new, complementary, DNA chains (also referred to as the developing strands) are separated from the template DNA
• The resulting single-stranded DNA chains are then separated according to length using gel electrophoresis

Question 6

How are single stranded lengths of DNA complementary to the template DNA separated?

Answer 6

gel electrophoresis

Question 7

What advancements have been made in sequencing

Answer 7

• 1986 - First automated DNA sequencing machine was developed.
• Using Fred Sanger’s method, fluorescent dyes were used to label the terminal bases instead of radioactive ones.
• These dyes then glowed when scanned with a laser beam, the light signature was identified by a computer.
• technicians were no longer required to read autoradiograms.

Question 8

what are the advantages of high- throughput sequencing?

Answer 8

were used to develop fast, cheap methods to sequence genomes

Question 9

give an example of High-throughput sequencing

Answer 9

pyrosequencing

Question 10

what’s pyrosequencing?

Answer 10

• Method that uses sequencing by synthesis, not by chain termination as in the Sanger method.
• massive parallel sequencing - can sequence many DNA at the same time.
• involves synthesising a single strand of DNA, complementary to the strand to be sequenced, one base at a time, whilst detecting by light emission, which base was added at each step

Question 11

what are the limits to chain termination sequencing

Answer 11

• chain termination method can only be used for DNA fragments up to 750 base pairs long
• to sequence entire genome of organism, DNA fragments need to be chopped into smaller pieces, sequenced, then put back together

Question 12

How are genomes sequenced

Answer 12

1. genome is cut into smaller fragments by restriction enzymes.
2. fragments inserted into bacterial artificial chromosomes (BACs) - these are man made plasmids. Each fragment is inserted into a different BAC.
3. BACs inserted into bacteria - each bacterium contains a BAC with a different DNA fragment.
4. bacteria divide, creating colonies of cloned cells that all contain a specific DNA fragment. Together the different colonies make a complete genomic DNA library.
5. DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA.
6. Each piece of DNA is sequenced, using the chain-termination method.
7. The pieces are put back in order to give the full sequence from that BAC (using powerful computer systems).
8. Finally, DNA fragments from all the BACs are put back in order by computers, to complete the entire genome.

Question 13

What’s bioinformatics?

Answer 13

• has grown out of the research from pyrosequencing.
• helps with the storage and analysis of large amounts of data.
• Software has been specifically designed to help with Bioinformatics

Question 14

applications of gene sequencing

Answer 14

• genome wide comparisons between species
• evolutionary relationships
• epidemiological studies
• to study phenotype-genotype relationships
• prediction a a sequence of proteins

Question 15

application of gene sequencing in terms of genome wide comparisons between species

Answer 15

• complete DNA of an organism’s genome determined
• eukaryotic cells = genetic material in chromosomes and mitochondria
• chloroplasts too in plants/algae

Question 16

application of gene sequencing in terms of comparison between species

Answer 16

• verifies that genes that work well tend are conserved by evolution
• many differences between organisms not because they have totally different genes but because shared genes are altered to work in different ways
• regulatory regions of DNA alter the expression of genomes
• helps build up picture of early human migration

Question 17

application of gene sequencing in terms of evolutionary relationships

Answer 17

• comparing genomes of organisms thought to be closely related has helped confirm evolutionary relationships

Question 18

application of gene sequencing in terms of epidemiological studies

Answer 18

• epidemiology - study of health and disease
• considers the distribution of a disease as well as its causes and effects
• some gene mutations linked to increased risk of disease
• computerised comparisons between genomes of ppl that have a disease and those that don’t can be used to detect particular mutations that increase risk

Question 19

application of gene sequencing in terms of genotype-phenotype relationships

Answer 19

• helps in the prediction of health problems as genotype is used to predict phenotype
• bioinformatics has allowed scientists to compare all the data and identity of genotype-phenotype relationships

Question 20

application of gene sequencing in terms of prediction aa sequence of proteins

Answer 20

• if genome has been sequenced, and the triplet code that codes for a specific protein is known then primary structure of protein can be determined
• researchers need to know parts of genes that code for introns and exons

Question 21

what’s synthetic biology?

Answer 21

• building biological systems from artificially made molecules (proteins) to see whether they work in the way we think they do
• redesigning biological systems to perform better and include new molecules
• redesign new biological systems and molecules that don’t exist in the natural world, but could be useful in humans

Question 22

what’s the difference between genetic engineering and synthetic biology

Answer 22

genetic engineering involves the direct transfer of DNA from one organism to another, whereas in synthetic biology, DNA is created from scratch