Sequencing methods

Question 1

DNA sequencing is the process of determining the —

enumerate the nucleotides

Answer 1

order of nucleotides

adenine, thymine, guanine, cytosine

Question 2

what are the two main types of DNA sequencing?

Answer 2

• Sanger sequencing
• next-generation sequencing

Question 3

used to terminate DNA synthesis in Sanger sequencing

Answer 3

dideoxynucleotides (ddNTPs)

Question 4

chain-termination method that uses ddNTPs to terminate DNA synthesis

Answer 4

Sanger sequencing

Question 5

dideoxynucleotides lack — which necessary for DNA synthesis to continue

Answer 5

3’-hydroxyl group

Question 6

how to perform sanger sequencing? enumerate.

Answer 6

1. DNA sample is amplified (PCR)
2. amplified DNA is incubated with a mixture of ddNTPs and dNTPs
3. ddNTPs will randomly terminate DNA synthesis, resulting in a series of DNA fragments of different lengths.
4. DNA fragments are separated by gel electrophoresis.
5. The order of the nucleotides in the DNA molecules is determined by reading the order of the bands on the gel.

Question 7

ddNTPs will randomly terminate DNA synthesis, what is the product?

Answer 7

series of DNA fragments of different lengths

Question 8

DNA fragments are then separated by — and the sequence of the DNA molecule is dtermined by?

Answer 8

separated by gel electrophoresis

sequence of DNA molecule is determined by reading the order of the bands on the gel

Question 9

collection of methods that can sequence DNA much faster and more cheaply.

Answer 9

next-generation sequencing

Question 10

this involve massively parallelizing the sequencing process

Answer 10

next-generation sequencing

DNA molecules are sequenced simultaneously

Question 11

DNA molecules are sequenced simultaneously. This is achieved by using a variety of techniques such as –

Answer 11

• sequencing by synthesis
• sequencing by ligation

Question 12

true or false

SBS can sequence entire genomes at a relatively low cost.

Answer 12

FALSE
next-generation sequencing

Question 13

NGS is used for —

Answer 13

• research
• diagnostics
• personalized medicine

Question 14

first DNA sequencing method and is widely used today. give the advantage and disadvantages of this method.

Answer 14

SANGER METHOD

advantages: simple and straightforward

disadvantage: time consuming and expensive to sequence large DNA molecules

Question 15

advantages of NGS

Answer 15

• can sequence large DNA molecules
• fast and cheap

Question 16

most common type of NGS method, it works by sequencing the DNA molecule on one nucleotide at a time.

Answer 16

sequencing by synthesis

Question 17

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?

Answer 17

sequencing by synthesis

Question 18

used to image the surface and determine the order of the nucleotides in the DNA molecules. this method refers to?

Answer 18

LASER

sequencing by synthesis

Question 19

sequencing the DNA molecule one fragment at a time.

Answer 19

sequencing by ligation

Question 20

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?

Answer 20

sequencing by ligation

Question 21

lecture

enumerate the importance of DNA sequence information

Answer 21

• detecting mutations
• typing microorganisms
• identify human haplotypes
• designating polymorphisms

Question 22

lecture

most specific esp when we use to identify genetic lesions, mutations, and polymorphisms

Answer 22

direct sequencing

Question 23

lecture

enumerate the 2 manual sequencing methods of direct

Answer 23

1. Chemical (Maxam-Gilbert) Sequencing
2. Dideoxy Chain termination (Sanger sequencing)

Question 24

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?

Answer 24

Chemical (Maxam-Gilbert) sequencing

Question 25

# **lecture** - manual sequencing a reducing agent which breaks the ssDNA at particular specific nucleotide of each base in each tube

Answer 25

10% pteridine

Question 26

# **lecture** enumerate the base modifiers and at what chain breaks at ---

Answer 26

1. dimetlysulphate = **G** 2. formic acid = **G + A** 3. hydrazine = **T + C** 4. hydrazine + salt = **C**

Question 27

# **lecture** this base modifier methylates G chain breaks at? | time (min @ 25C)?

Answer 27

**Dimethylsulphate** breaks at **G** | 4 @25 C

Question 28

# **lecture** this base modifier **protonates purine** and chain *breaks at G+A*

Answer 28

formic acid | 5 at 25C

Question 29

# **lecture** **splits pyrimidine rings** and chain *breaks at T+C*

Answer 29

hydrazine

Question 30

# **lecture** this base modifier splits only C rings, chain breaks at C

Answer 30

hydrazine + salt

Question 31

# **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.

Answer 31

Chemical (Maxam-Gilbert) sequencing

Question 32

# **lecture** there is the addition of a nucleotide triphosphate - which requires a free hydroxyl group from 3' carbon. this method refers to?

Answer 32

Dideoxy Chain termination **Sanger sequencing**

Question 33

# **lecture** - manual sequencing strand synthesis reaction is catalyzed by a type of DNA polymerase known as?

Answer 33

**Klenow fragment**

Question 34

# **lecture** method of sanger sequencing

Answer 34

1. anneal a short oligonucleotide (17-24 nucleotides), to the same position on each molecule. 2. ddTTP is then added to stop the reaction. 3. perform our DNA synthesis in 4 separate tubes 4. perform electrophoresis under denatured condition

Question 35

# **lecture** - sanger sequencing act as a substrate that in the 4 tubes

Answer 35

4 dideoxynucleotides 4 deoxyribonucleotide triphosphates

Question 36

# **lecture** products of the four-sequencing reaction

Answer 36

sequencing ladder

Question 37

# **lecture** this is performed to visualize the DNA fragments which will appear as horizontal bands on an x-ray film.

Answer 37

**AUTORADIOGRAPHY** | it is performed bcos of radioactive ddATP is included in your reaction.

Question 38

# **lecture** sequencing enzyme which has *low processivity* means that it can **only synthesize short DNA strands** before dissociating from the template

Answer 38

Klenow polymerase

Question 39

# **lecture** drawback of klenow polymerase

Answer 39

limit the length of the sequence tjat can be obtained from a single experiment to only **250 base pairs**

Question 40

# **lecture** modified version of the DNA polymerase that is encoded by **bacteriophage T7**

Answer 40

sequenase | to improve short synthesis problem

Question 41

# **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?

Answer 41

sequenase | **750 base pairs** in just a single experiment

Question 42

# **lecture** use to inset our DNA and only good to use if your DNA fragments are **shorter than 3kb**

Answer 42

M13 vector

Question 43

# **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**

Answer 43

**PLASMID VECTOR** alternative for M13

Question 44

# **lecture** most common method for obtaining a template DNA for DNA sequencing

Answer 44

**DENATURATION** adding of alkali | boiling

Question 45

# **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

Answer 45

phagemid | **more than 10 kb** DNA fragments

Question 46

# **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**

Answer 46

automated method

Question 47

# **lecture** - automated method how can we separate the moleculres according to their lengths after the extension reaction?

Answer 47

polyacrylamide gel | capillary gel electrophoresis

Question 48

# **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?

Answer 48

**1:1** * **high** = polymerization will terminate to frequently early on the template * **low** = no termination that will occur

Question 49

# **lecture** the reactions are terminated by addition of a **stop buffer**, this contains ---

Answer 49

- 20 mM EDTA - formamide - gel loading dyes

Question 50

# **lecture** used to chelate cations and stop enzyme activity

Answer 50

20 mM EDTA

Question 51

# **lecture** this will denature the product of the synthesis reaction

Answer 51

formamide

Question 52

# **lecture** what are the gel loading dyes?

Answer 52

- bromophenol blue - xylene cyanol

Question 53

# **lecture** first of the high throughput DNA sequencing or next generation sequencing technology. this is for large amount of data about genomic DNA

Answer 53

pyrosequencing

Question 54

# **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.

Answer 54

pyrosequencing

Question 55

# **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.

Answer 55

pyrosequencing

Question 56

# **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 ---

Answer 56

conversion of **luciferen to oxyluciferin** by the enzyme luciferase

Question 57

# **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.

Answer 57

**oxyluciferin**

Question 58

# **lecture** light is --- to the amount of pyrophosphate produced and --- to the number of nucleotides added

Answer 58

DIRECTLY PROPORTIONAL

Question 59

# **lecture** catalyzes the degregation of the excess dNTPs before we add next dNTP

Answer 59

apyrase

Question 60

# **lecture** advantages of pyrosequencing

Answer 60

- rapid - can be automated - does not require electrophoresis - hundreds of thousands of sequences can be obtained

Question 61

# **lecture** sequence assembly can be done through?

Answer 61

- shotgun approach - clone contig approach

Question 62

# **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

Answer 62

shotgun cloning approach | this is only done for **smaller and shorter bacterial genome**

Question 63

# **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?

Answer 63

clone contig approach | **contig** - overlapping clones

Question 64

# **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

Answer 64

5' single stranded = **polymerase** 3' = **exonuclease**

Question 65

# **lecture** vector used in shotgun cloning which will act as a sequencing template

Answer 65

E. coli

Question 66

# **lecture** this approach provides an accurate sequence of a large genome that contains repetitive DNA | what is the disadvantage?

Answer 66

clone contig approach | time consuming and requires effort

Question 67

# **lecture** enumerate the high capacity vectors

Answer 67

1. bacterial artificial chromosome (BAC) 2. yeast artificial chromosome (YAC)

Question 68

# **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?

Answer 68

600 - 700 base pairs | pair end

Question 69

# **lecture** enumerate the techniques used in clone contig assembly (4)

Answer 69

- chromosome walking - clone fingerprinting - interspersed repeat element PCR - sequence tagged site content analysis

Question 70

# **lecture** this technique is used to move relatively short distances along DNA molecules, using clone libraries prepared with **lambda or cosmind vector**

Answer 70

chromosome walking

Question 71

# **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**

Answer 71

**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**

Question 72

# **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. ## Footnote the simplest approach is to digest each clone with a variety of restriction endonucleases.

Answer 72

clone fingerprinting

Question 73

# **lecture** it uses primers that are design to anneal within repetitive DNA sequences and direct amplification of the DNA between adjacent repeats.

Answer 73

interspersed repeat element PCR (IRE-PCR)

Question 74

# **lecture** this can be used as a fingerprint in comparison to the other clones in order to identify potential overlaps.

Answer 74

DNA PCR

Question 75

# **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.

Answer 75

sequence tagged site content analysis

Question 76

# **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?

Answer 76

sequence tagged site

Question 77

# **lecture** only requirement of sequence tagged site

Answer 77

it must occur at **once only in the gene**