AMB7 Flashcards
(15 cards)
Describe Maxam Gilbert Sequencing
first generation sequencing.
Radiolabel the 5’ end of the DNA
chemically treat it to induce breaks between one or two of the four nucleotides in each of four reactions, G, A+G, T+C and C.
- formic acid depurinates the purines (A+G)
- hydrolysis of pyrimidines
- dimethyl sulfate methylates guanine.
add hot piperidine to cleave the DNA at these modifications
Left with fragments of DNA with random lengths.
Electrophoresis for size seperation and autoradiography, can infer sequence by presence or absense of bands.
Describe first gen enzymatic sanga sequencing
template molecule is copied using oligonucletide primer and DNAP
four test tubes containing each for dNTPs plus one of each type of ddNTP in each test tube
reactions terminated at random but at points specifically identified by the terminator ddNTPss used
High resolution Electrophoresis and comparisons to determine sequence
what is a ddNTP
di-deoxy nucletodie = 2, 3. - dideoxy as no 3’OH to bond
what is the processivity of an enzyme
measure of how many bases are incorperated by enzyme before it falls fof
Describe Sanga seqeuncing cycle sequencing
dsDNA used and denatured at 95 degrees
one oligonucletodie primer (can be labelled) present anneals at 50 degrees
primer extended using taq polymerase in presence of dNTPs and ddNTPs (72 degrees)
Get products that are only partial products
seperated by denturing and used again.
electrophoresis of different size fragements
Where does illumina sequencing take place
on a flow cell, a glass slide with lanes contining a lawn of two types of single stranded oligonucleotides bound.
what is clustering
process by which each fragment is isothermally amplified
Describe the first stage of illumina sequencing by synthesis
- DNA to be sequenced is cleaved into random fragments and adaptors ligated to each end.
- Fragment passes over lawn and one end hybridises with complimentary first oligonucletide
- DNAP extends using first oligonucletodie as a primer
- once extended ds is denatured and template strand is washed away
- clonal amplification then occurs through bridge amplification whereby the strand bends and adaptor on the other end hybridises to second oligonucleotide
- DNAP then makes a double stranded bridge
- This is then denatured to form two single strands which bind to complementary adaptors continualling the bridge amplification. This repeated over and over again, simultaneously for all the fragments
- end up with a cluster of up to a million clones of the fragment.
- The reverse strand is then washed away leaving on the forward strand and the 3’ end is blocked to prevent undwanted priming.
Describe the second stage of illumina sequencing by synthesis
- sequencing begins with extention of first primer to produce the first read.
- with each sequence fluorescently tagged nts compete for addition to growing chain
- only one incorporated on template sequence, after each addition bases are excited by a light fluoresecent signal and a characteristic signal is admited.
- no. oc cyles determined by length of read.
- for a given cluster all identical strands are sequenced simultaneously.
- all fragments are sequenced in parallel
- reads are repeated and fragments compared to find order.
what is metagenomics
sequencing of genomes of a community of organisms inhabiting a common environment.
Describe 3rd generation pac bio sequencing.
cell has tiny pores at the bottom, DNAP bound at the base of the pore when DNA feeds into the well DNAP starts polymerising compliment
Optical behaviour at the bottom of the well changes so can measure which base is added in a single molecule using wave guide phenomenon.
What is the Zero Mode Wave guide
A zero-mode waveguide is an optical waveguide that guides light energy into a volume that is small in all dimensions compared to the wavelength of the light.
What is the size of the well in 3rd generation pac bio sequencing and why
10 and 10 ^21 L allows direct detection of activity by detection of light behaviours in volumes that are int he order of magnitude or smaller than the wavelengths.
What are the advantages of 3rd gen pac bio sequencing
- one molecule dna used not clone like illumina
- efficient and can sequence much longer strands of dna up to 40-50 kbs
- overcomes the problem of trying to ordering where repeats are in DNA
- errors in the outputs are randomly distributed so repeated sequencing can detect effectively which sequnces are mistaken adn correct mistakes
what are the disadvantages of pac bio sequencing
accuracy is relatively low, higher in illumina. - can be soved by repeats.
not so much higher througput but helps in process of assembling genome.
