Methods 2 (lecture 4) Flashcards
If you want to subclone a 2.5kb DNA fragment from genomic DNA that does not have suitable restriction sites (i.e., none flanking the insert or compatible with the multiple cloning site of the vector you need to use), how will you solve this problem and make the construct?
PCR amplify the fragment, design primers w/ extensions on the 5’ ends that contain restriction sites (~10nt) and match the DNA of interest on the 3’ ends (~20nt), use proofreading DNA polymerase for PCR, digest with appropriate restriction enzymes, ligate plasmid and fragment together, transform e. coli
What is a proofreading DNA polymerase
Proofreading DNA polymerase in PCR is an enzyme that can detect, remove, and replace incorrect bases in DNA during replication. This process is also known as 3’-5’ exonuclease activity.
Types of 1st gen DNA sequencing (2 types)
- Maxam-Gilbert chemical modification and cleavage (outdated)
- Sanger dideoxy chain-termination (relevant)
Types of next-gen or 2nd generation or massively parallel sequencing (4 types)
- 454 pyrosequencing, roche
- Illumina (Solexa) sequencing by synthesis
- SOLiD sequencing by ligation, life technologies (Applied Biosystems)
- Ion Torrent semiconductor, life technologies
Steps of Maxam-Gilbert sequencing
- 5’-ends of dsDNA are radioactively labeled by polynucleotide kinase and [g-32P]-ATP
- dsDNA is denatured in the presence of DMSO and heat, and ssDNA is purified*
- Labeled ssDNA is split into 4 reactions, chemically treated and cleaved after specific bases
- Obtained fragments are separated on a polyacrilamide gel and autoradiographed
Single-molecule or 3rd generation sequencing (3 types)
- Helicos Biosciences
- Single Molecule Real Time (SMRT), Pacific Biosciences
- Oxford Nanopore
In maxam-gilbert sequencing how is ssDNA purified?
centrifugation, one strand will have more purines (larger) one more pyrimidines (smaller)
In maxam-gilbert sequencing why is radiolabeled ssDNA split into 4 reactions
Different chemicals are used which break the bonds between different nucleotides, by running 4 seperate reactions on the same gel you can get base pair resolution of you DNA
Why do ssDNA need to be separated by size in the Maxam-Gilbert method?
I the both complementary strands are run on the gel you will not be able to tell which fragments belong to which strand as both will be bottom -> top or gel starting from labeled 5’ end
What is the name of the fluorescent nucleotide terminator utilized in Sanger sequencing?
fluorescently labeled dideoxynucleotide
What are the ingredients for sanger sequencing reaction
DNA polymerase I
Primer
dATP
dGTP
dCTP
dTTP
+limiting amounts of fluorescently labeled:
ddATP
ddGTP
ddCTP
ddTTP
What is the full name of sanger sequencing?
Sanger chain termination (dideoxy) method
Difference between dideoxynucleotide and deoxynucleotide?
dideoxynucleotide lack 3’-OH (-OH on the 3’ carbon of ribose) which prevent the next nucleotide from attaching, DNA synthesis will terminate
How can dideoxynucleotides be used to treat diseases?
azidothymidine is used to treat ends, HIV prefers AZT to normal thymidine (dTTP), DNA synthesis halts as AZT has no 3’ -OH group, side effects are mild on host cell as they prefer dTTP
What is done with dideoxylabeled ssDNA fragments in sanger sequencing?
- DNA is seperated by size in an electric field (polyacrylamide gel or capillary) using electrophoresis,
- as fluorescently labeled fragments of discrete sizes pass a detector they are activated by a laser and read by a photometer
- Read from bottom of gel to top, you will be reading the complement to your original sequence
What did the original sanger method use to label dideoxynucleotides?
radioactivity, would use 4 separate reactions for each nucleotide
What will happen if a heterozygote gene is sequenced using sanger sequencing?
there will be 50/50 color split of 2 different bps in same spot
what determines which strand is read in sanger sequencing?
the primer design
How many bp resolution is sanger sequencing?
up to 1000 bp, any longer and you must design an internal primer
how do you amplify DNA for sanger sequencing?
fragment DNA, subclone into a vector, amplify in vivo, then sequence the vector
How is next-gen sequencing similar/different from sanger?
Both start with DNA fragmentation, next-gen adds adaptors, uses PCR to make a form of spatially immobilized PCR colonies containing many copies of DNA fragments (features), detectors can acquire sequencing data on all features in parallel
454 pyrosequencing is sequencing by ______
synthesis
454 pyrosequencing utilizes a series of _________ to detect _________ released during _________
enzymatic reactions
pyrophosphate (PPi)
base incorporation
what is the function of apyrase in 454 pyrosequencing
hydrolyzes/inactivates extra nucleotides (not incorporated in the DNA)
what is the function of Luciferase in 454 pyrosequencing
hydrolyzes ATP in the presence of its substrate luciferin, producing oxyluciferin and light
what is the function of Sulfurylase in 454 pyrosequencing
regenerates ATP (out of APS (adenosine 5′-phosphosulfate) and PPi)
what is the function of DNA polymerase in 454 pyrosequencing
adds nucleotides and releases PPi
name of the figure/graph 454 outputs to tell you nucleotide sequence
pyrogram, each peak represent a pulse of light, twice as big if m
explain SOLiD sequencing by ligation simply
- begins like 454 pyro, emulsion PCR is performed on beads covered in adaptor sequences and (ideally) 1 DNA fragment/bead
- PCR fragments are modified on 3’ end to covalently attach beads + fragments to a glass slide
- 16 interrogation probes are added 4 at times, octomers (3’ –> 5’) 2 probe specific bases, 6 degenerate bases, fluorescent label on 5’ end
- adaptor sequences on the bead bind a universal primer, the primer is the starting point from where probes will ligate.
- 2 bases/5 are interogated up the length of the template, when base binds the fluorescent end is cleaved
- the extenstion product is removed and template reset with a primer complementary to one position over from previous primer is attached, repeat the ligation cycles 5x
how many times is each base interrogated in SOLiD sequencing?
2 times
what chemical cleaves the fluorescent label from SOLiD sequencing probes and what is the type of bond cleaved?
AgNO3 is the chemical which cleaves the phosphorothioate bond
Illumina is sequencing by _______
synthesis
explain library construction for illumina sequencing
- genomic DNA or cDNA is fragmented and adaptors are added
- adaptor sequences allow hybridization to the flow cell and also contain a binding site for the illumina sequencing primer to bind
- PCR amplifies genomic DNA, primers bridge amplification results in clusters of DNA
- labeled ddNTPs are added (containing dye and a terminator group)
- wash and image, you now know the next nuc in the sequence
- cleave the dye and terminator group and repeat
why is it important that sanger and illumina sequencing first make clusters of DNA?
clusters = stronger signal as nucleotides are added making it easier for a machine to read
All NGS sequencing approaches enable _____
parallel sequencing of millions of independent fragments
ion torrent semiconductor sequencing is sequencing by ____
synthesis
where does the H+ released during ion torrent sequencing come from?
the hydroxyl group of the remaining phosphate attached to the base, pyrophosphate (2 phosphates) are cleaved and the new nucleotide attaches in the place of the H+
steps of ion torrent semiconductor sequencing?
- library prep (fragment DNA, add adaptors, attach to beads)
- PCR amplification
- nucleotides are incorporated one at a time, if the nucleotides are incorporated H+ protons are released
- the pH changes when H+ is released and this is sensed by the machine as an electrical signal
what NGS sequencing technologies are not great to sequence long strings of repetitive bases?
ion torrent, 454 pyrosequencing,
SMRT stands for what
single molecule real time
SMRT is sequencing by _______
synthesis
how does SMRT sequencing work?
- 1 molecule/ well of dsDNA has SMRTbell adaptors added (hairpin adaptor that makes circular template)
- primers added to adaptor and DNA polymerases binds primer
- DNA polymerase incorporates labeled nuceotides
what is the name of the well which DNA polymerase enzymes are affixed during SMRT sequencing?
zero-mode waveguide (ZMW) well, this structure creates an illuminated observation volume small enough to observe single nucleotides being attached by a fluorescence detector
How does SMRT handle being low fidelity?
strands are read multiple times as dsDNA was linearized, the consensus sequence is more accurate
what sequencing technology is good for long read?
pacbio SMRT and oxford nanopore (3rd gen) produce reads tens of thousand nt long
simple explanation of how 1st, 2nd, and 3rd gen seq work, fragment size they read, names of seq tech
first gen (sanger, maxam and gilbert): infor nucleotide if with dNTPs then visualize with electrophoresis, 500-1000 bp fragments
second gen (454, illumina, solexa, ion torrent): high throughput from parallelization of sequencing reads, 50-500 bp fragments
third gen (Pacbio SMRT, oxford nanopore): sequence native DNA in real time with single molecule resolution, tens of kb fragment reads
Do Pacbio and oxford nanopore include PCR amplification step?
No
