DNA Sequencing Flashcards
(65 cards)
Define the term DNA sequencing
-a technique that allows genes to be isolated and read
What are the 2 key methods for DNA sequencing
1) Sanger sequencing
2) Pyrosequencing
What are the 5 ingredients needed for Sanger sequencing
1) DNA polymerase enzyme
2) primer
3) four DNA nucleotide
4) template DNA to be sequenced
5) dideoxynucleotide
What is DNA polymerase enzyme used for in Sanger sequencing
-to catalyse the addition of the free nucleotides
What is a primer and what is its role in Sanger sequencing
-a short piece of single-stranded DNA
-its role is to bind to the template DNA and act as a “starter” for the polymerase
What is the role of the dideoxynucleotides
-they are chain terminating versions of the four nucleotides and end the sequencing when present
How come dideoxynucleotides end sequencing
-because they lack an -OH group on carbon 3 so they don’t allow for replication to be continued
Explain the method of Sanger sequencing
1) ingredients are added to a tube
2) the mixture is heated up (to separate the phosphodiester bonds between the strands)
3) it is then cooled so that the primer can bind to the single-stranded template
4) DNA polymerase synthesises new DNA starting from the primer
5) DNA polymerase continues adding nucleotide to the chain until it happens to add a dideoxynucleotide instead of a normal one, so no further nucleotides can be added so the strand ends with the dideoxynucleotide
6) process repeats itself in number of cycles
7) the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA
8) the ends of the fragments will be labelled with radioactive primer
9) these fragments are then sorted into size order by electrophoresis
What does the Sanger sequence ensure
-that a dideoxynucleotide will have been incorporated at every single position of the target DNA in at least one reaction
What does the Sanger sequencing method rely on
-the chain terminating (dideoxynucleotides) being incorporated
What is a disadvantage of Sanger sequencing
-it is very slow
Explain cloning DNA using a vector
1) GENE ISOLATION
->specific gene from a bacterium is cut out using restriction enzymes, which recognise and cut specific DNA sequences
2) INSERTION INTO A VECTOR
->the isolated gene is inserted into a bacterial plasmid (vector)
3) TRANSFORMATION INTO A HOST BACTERIA
-> the genetically modified plasmid is introduced into E. coli
4) BACTERIAL REPLICATION AND GENE AMPLIFICATION
->as the E. coli cells divide they replicate the plasmid, making many copies of the inserted gene
5) PLASMID ISOLATION
->the bacteria are cultured and the plasmids containing the gene of interest are extracted using plasmid preparation techniques
6) DNA SEQUENCING
->the isolated DNA is then sequenced to determine its nucleotide order which allows researchers to analyse the genes structure and function
What does pyrosequencing use on its terminal bases compared to Sanger sequencing
-pyrosequencing uses fluorescent dyes instead of radioactivity
How can scientist identify terminal bases in pyrosequencing
-they scan strands with laser beam and the dyes glow, the light signature was identified by computer
What piece of equipment is used to read pyrosequences
-autoradiograms
How does pyrosequencing use sequencing
-by synthesis
Summarise what pyrosequencing 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
What are the 9 ingredients needed in pyrosequencing
1) DNA sample
2) primer
3) DNA polymerase
4) modified nucleotides
5) ATP sulfurylase
6) luciferase
7) apyrase
8) luciferin
9) APS (adenosine phosphosulfate)
What is ATP sulfurylase
-an enzyme that forms ATP from adenosine phosphosulfate (APS) and pyrophosphate (PPi)
What is luciferase
-an ATPase that catalyses the conversion
What is apyrase
- removes unincorporated nucleotides (nucleotides that were not added to the growing DNA strand during DNA synthesis)
What is luciferin
-a generic term for the light-emitting compound found in organisms that generate bioluminescence
Explain the method of pyrosequencing
1) long piece of DNA is cut into fragments using a nebuliser
2) these lengths are degraded to single stranded DNA
3) all ingredients are added- but only 1 of the 4 possible activated nucleotides is added at any one time
4) when a modified nucleotide is added, the two extra phosphorylase are released as pyrophosphate (PPi)
5) in the presence of APS, the enzyme ATP sulfurylase converts PPi to ATP
6) in the presences of ATP, the enzyme luciferase converts luciferin to oxyluciferin which generates visible light that is detected by a camera
7) the amount of light generated is proportional to the amount of ATP available and therefore indicated how many of the same type of activated nucleotides were incorporated adjacently
8) this is recorded on a pyrograph
9) unincorporated nucleotides are degraded by apyrase
What size of DNA can be sequenced in Sanger sequencing
Large
