Sequencing methods Flashcards
DNA sequencing is the process of determining the —
enumerate the nucleotides
order of nucleotides
adenine, thymine, guanine, cytosine
what are the two main types of DNA sequencing?
- Sanger sequencing
- next-generation sequencing
used to terminate DNA synthesis in Sanger sequencing
dideoxynucleotides (ddNTPs)
chain-termination method that uses ddNTPs to terminate DNA synthesis
Sanger sequencing
dideoxynucleotides lack — which necessary for DNA synthesis to continue
3’-hydroxyl group
how to perform sanger sequencing? enumerate.
- DNA sample is amplified (PCR)
- amplified DNA is incubated with a mixture of ddNTPs and dNTPs
- ddNTPs will randomly terminate DNA synthesis, resulting in a series of DNA fragments of different lengths.
- DNA fragments are separated by gel electrophoresis.
- The order of the nucleotides in the DNA molecules is determined by reading the order of the bands on the gel.
ddNTPs will randomly terminate DNA synthesis, what is the product?
series of DNA fragments of different lengths
DNA fragments are then separated by — and the sequence of the DNA molecule is dtermined by?
separated by gel electrophoresis
sequence of DNA molecule is determined by reading the order of the bands on the gel
collection of methods that can sequence DNA much faster and more cheaply.
next-generation sequencing
this involve massively parallelizing the sequencing process
next-generation sequencing
DNA molecules are sequenced simultaneously
DNA molecules are sequenced simultaneously. This is achieved by using a variety of techniques such as –
- sequencing by synthesis
- sequencing by ligation
true or false
SBS can sequence entire genomes at a relatively low cost.
FALSE
next-generation sequencing
NGS is used for —
- research
- diagnostics
- personalized medicine
first DNA sequencing method and is widely used today. give the advantage and disadvantages of this method.
SANGER METHOD
advantages: simple and straightforward
disadvantage: time consuming and expensive to sequence large DNA molecules
advantages of NGS
- can sequence large DNA molecules
- fast and cheap
most common type of NGS method, it works by sequencing the DNA molecule on one nucleotide at a time.
sequencing by synthesis
DNA molecule is first attached to a solid surface. then a mixture of nucleotides is added to the surface. the nucleotides will bind to the complementary nucleotides in the DNA molecule. this method refers to?
sequencing by synthesis
used to image the surface and determine the order of the nucleotides in the DNA molecules. this method refers to?
LASER
sequencing by synthesis
sequencing the DNA molecule one fragment at a time.
sequencing by ligation
DNA molecule is first fragmented into small pieces then the fragments are attached to adapters. the adapters contain sequences that are known to bind to the sequencing machine. the fragments are then ligated together to form a long chain. the sequencing machine then reads to the order of the nucleotides in the chain. this method refers to?
sequencing by ligation
lecture
enumerate the importance of DNA sequence information
- detecting mutations
- typing microorganisms
- identify human haplotypes
- designating polymorphisms
lecture
most specific esp when we use to identify genetic lesions, mutations, and polymorphisms
direct sequencing
lecture
enumerate the 2 manual sequencing methods of direct
- Chemical (Maxam-Gilbert) Sequencing
- Dideoxy Chain termination (Sanger sequencing)
lecture
the labeled fragment or template is aliqouted into 4 tubes and each of this tubes contain a base modifier, this refers to what method?
Chemical (Maxam-Gilbert) sequencing
lecture - manual sequencing
a reducing agent which breaks the ssDNA at particular specific nucleotide of each base in each tube
10% pteridine
lecture
enumerate the base modifiers and at what chain breaks at —
- dimetlysulphate = G
- formic acid = G + A
- hydrazine = T + C
- hydrazine + salt = C
lecture
this base modifier methylates G
chain breaks at?
time (min @ 25C)?
Dimethylsulphate
breaks at G
4 @25 C
lecture
this base modifier protonates purine and chain breaks at G+A
formic acid
5 at 25C
lecture
splits pyrimidine rings and chain breaks at T+C
hydrazine
lecture
this base modifier splits only C rings, chain breaks at C
hydrazine + salt
lecture
this method is very direct way to sequence DNA but **not practical for high throughput ** esp. for long fragments because this is tedious to do.
Chemical (Maxam-Gilbert) sequencing
lecture
there is the addition of a nucleotide triphosphate - which requires a free hydroxyl group from 3’ carbon. this method refers to?
Dideoxy Chain termination
Sanger sequencing
lecture - manual sequencing
strand synthesis reaction is catalyzed by a type of DNA polymerase known as?
Klenow fragment
lecture
method of sanger sequencing
- anneal a short oligonucleotide (17-24 nucleotides), to the same position on each molecule.
- ddTTP is then added to stop the reaction.
- perform our DNA synthesis in 4 separate tubes
- perform electrophoresis under denatured condition
lecture - sanger sequencing
act as a substrate that in the 4 tubes
4 dideoxynucleotides
4 deoxyribonucleotide triphosphates
lecture
products of the four-sequencing reaction
sequencing ladder
lecture
this is performed to visualize the DNA fragments which will appear as horizontal bands on an x-ray film.
AUTORADIOGRAPHY
it is performed bcos of radioactive ddATP is included in your reaction.
lecture
sequencing enzyme which has low processivity means that it can only synthesize short DNA strands before dissociating from the template
Klenow polymerase
lecture
drawback of klenow polymerase
limit the length of the sequence tjat can be obtained from a single experiment to only 250 base pairs
lecture
modified version of the DNA polymerase that is encoded by bacteriophage T7
sequenase
to improve short synthesis problem
lecture
this has high processivity that can synthesize longer strands compared to the klenow polymerase and NO EXONUCLEASE ACTIVITY making it ideal for chain termination sequencing
how many base pairs can be sequenced in just a single expirement?
sequenase
750 base pairs in just a single experiment
lecture
use to inset our DNA and only good to use if your DNA fragments are shorter than 3kb
M13 vector
lecture
this vector is needed to convert the double-stranded plasmid into a single-stranded form through the addition of an alkali or through boiling
PLASMID VECTOR
alternative for M13
lecture
most common method for obtaining a template DNA for DNA sequencing
DENATURATION
adding of alkali | boiling
lecture
a plasmid vector that contains an M13 origin of replication and which can be obtained as both a double- or single-stranded DNA version
this can be used — of DNA fragments
phagemid
more than 10 kb DNA fragments
lecture
each of the dideoxynucleotide used in the reaction is labeled with a different fluoresent marker, this method refers to?
termination sequencing is only performed in one tube
automated method
lecture - automated method
how can we separate the moleculres according to their lengths after the extension reaction?
polyacrylamide gel | capillary gel electrophoresis
lecture
ratio of ddNTPs and dNTPs
this is important for the generation of a readable sequence
what is the effect if high concentration of ddNTPs or if it is too low?
1:1
- high = polymerization will terminate to frequently early on the template
- low = no termination that will occur
lecture
the reactions are terminated by addition of a stop buffer, this contains —
- 20 mM EDTA
- formamide
- gel loading dyes
lecture
used to chelate cations and stop enzyme activity
20 mM EDTA
lecture
this will denature the product of the synthesis reaction
formamide
lecture
what are the gel loading dyes?
- bromophenol blue
- xylene cyanol
lecture
first of the high throughput DNA sequencing or next generation sequencing technology. this is for large amount of data about genomic DNA
pyrosequencing
lecture
can be automated in a massively parallel manner that neables hundreds of throusands of sequences that can be obtained at once or has even at a hundred million of base pairs in just a single run.
pyrosequencing
lecture
the basis of — is the detection of pyrophosphate that is released during DNA synthesis. it requires preparation first of an identical single-stranded DNA molecule.
pyrosequencing
lecture - pyrosequencing
pyrophosphate will then combine with adenosine-5’-phosphosulfate in the presence of the enzyme ATP sulfurylase to form ATP. this ATP would drive the conversion of — by the enzyme —
conversion of luciferen to oxyluciferin by the enzyme luciferase
lecture
— will then generate light. the amount of light is detected after each cycle of nucleotide addition and the enzymatic reaction indicates the incorportaion of a complementary nucleotide.
oxyluciferin
lecture
light is — to the amount of pyrophosphate produced and — to the number of nucleotides added
DIRECTLY PROPORTIONAL
lecture
catalyzes the degregation of the excess dNTPs before we add next dNTP
apyrase
lecture
advantages of pyrosequencing
- rapid
- can be automated
- does not require electrophoresis
- hundreds of thousands of sequences can be obtained
lecture
sequence assembly can be done through?
- shotgun approach
- clone contig approach
lecture
this sequence assembly will break down the genome randomly into short fragments. it will then be used to build-up through seeing which sequence will fit unto the length of the DNA
shotgun cloning approach
this is only done for smaller and shorter bacterial genome
lecture
a presequencing phase during which a series of overlaping clones is identified. longer lengths which of overlaps twith each other.
overlapping clones are called?
clone contig approach
contig - overlapping clones
lecture - shotgun cloning
if it ends in the 5’ single stranded end, — is used to fill in the 3’ recessed end of the complementary strand.
if it ends on the 3’ single stranded end, — is used to remove protruding end.
library - uniformly sized DNA fragments
5’ single stranded = polymerase
3’ = exonuclease
lecture
vector used in shotgun cloning which will act as a sequencing template
E. coli
lecture
this approach provides an accurate sequence of a large genome that contains repetitive DNA
what is the disadvantage?
clone contig approach
time consuming and requires effort
lecture
enumerate the high capacity vectors
- bacterial artificial chromosome (BAC)
- yeast artificial chromosome (YAC)
lecture - clone contig
each fragment is cloned in a vector and is sequenced from both ends to produce a sequence length of — base pairs
sequnce of both ends through the DNA fragment is called?
600 - 700 base pairs
pair end
lecture
enumerate the techniques used in clone contig assembly (4)
- chromosome walking
- clone fingerprinting
- interspersed repeat element PCR
- sequence tagged site content analysis
lecture
this technique is used to move relatively short distances along DNA molecules, using clone libraries prepared with lambda or cosmind vector
chromosome walking
lecture
this is a technique where you are attempting to move short molecules through the vector
what could be the weakness for this technique?
aim: close one or more small gaps between contigs
CHROMOSOME WALKING
weakness: slow process can only assemble at aroung 15-20 clones
this is only attempted when the contig is for a short chromosome
lecture
this is the identification of sequence features that are shared by a pair of clones. it also provide information on a physical structure of a cloned DNA fragment.
the simplest approach is to digest each clone with a variety of restriction endonucleases.
clone fingerprinting
lecture
it uses primers that are design to anneal within repetitive DNA sequences and direct amplification of the DNA between adjacent repeats.
interspersed repeat element PCR (IRE-PCR)
lecture
this can be used as a fingerprint in comparison to the other clones in order to identify potential overlaps.
DNA PCR
lecture
this is when we search for pairs of clones that contain a specific DNA sequence that occurs at just one position in the genome under study.
sequence tagged site content analysis
lecture
if two clones contain a specific DNA sequence that occurs at just one position in the genome must overlap. a sequence of this type is called?
sequence tagged site
lecture
only requirement of sequence tagged site
it must occur at once only in the gene
