sequencing, vectors, gene libraries lecture 7 and 8

Question 1

Large volume of DNA Fragments Can be rapidly sequenced due to the development in the mid-1970s of….

Answer 1

The dideoxy method for sequencing DNA

Question 2

what is the dideoxy method for sequencing DNA based on

Answer 2

it is based on in vitro DNA synthesis performed in the presence of chain-terminating dideoxyribonucleoside triphosphates (ddNTPs)

Question 3

The Sanger method

Answer 3

• invented by Sanger, Smith & Coulson in mid 1970s, it is based on synthesis of a complementary DNA strand by DNA polymerase, initiated by a short oligonucleotide primer, only uses one primer and one template strand.
  Also called:
• The dideoxy method
• The chain termination method

Question 4

What does incorporation of dideoxynucleotides leads to in DNA sequencing?

Answer 4

chain termination

Question 5

dideoxyribonucleoside triphosphates is a derivatives of normal deoxyribonucleoside triphosphates but lacks the…

Answer 5

3′ hydroxyl group.

Question 6

What will happen if you mix some di-deoxy-NTPs into a sequencing reaction?

Answer 6

DNA polymerase cannot elongate DNA strand without 3’ hydroxyl group therefore it prevents strand extension at 3’ end

Question 7

What steps are involved in Sanger Sequencing

Answer 7

• The dsDNA fragment of interest is denatured to form a single stranded (ssDNA) template
• The ssDNA is added to a solution (gene of interest, DNA polymerase, All four dNTPs, small quantity of a single ddNTP. (This step is done for each of the four nucleotides in separate solutions)
• The normal dNTP will usually be incorporated, but occasionally, at random, the ddNTP will be incorporated and the reaction will stop
• In each tube, DNA fragments will be synthesised that terminate at a ddNTP location, producing fragments of varying lengths

Question 8

What happens when the reaction is complete

Answer 8

• each solution containing a specific dNTP and ddNTP are placed in their own lane of a gel and run under gel electrophoresis
• The gel is transferred to a polymer sheet and autoradiography is used to visualise the bands in the gel
• The sequence can be determined by reading down the position of the fragments in the gel, giving the complementary sequence to the original ssDNA template

Question 9

Advantages and Limitations of Sanger sequencing

Answer 9

• Sanger sequencing reaction can be purchased for ~10€ per run, making it very cheap and accessible.
• It’s the method of choice for verifying DNA constructs, PCR products etc.
  However:
• It requires relatively large amounts of template.
• Only sequences up to 1kB can be reliably generated by Sanger sequencing.
• If longer sequences are required, several sequencing runs with different sequencing primers are required (sequence overlapping fragments).

Question 10

Overlapping fragments- ‘primer walking’

Answer 10

• Start sequencing a large DNA fragment.
• Use the initial sequencing result (~750bp long) to design primer for second sequencing reaction and so on…
• Eventually you will have sequenced the whole fragment.
• Using this technique, you keep track of how the individual ‘reads’ align together.

Question 11

what is the alternative to primer walking’

Answer 11

the shotgun approach

Question 12

what is the the shotgun approach

Answer 12

Use restriction enzymes or ultrasound to break the DNA into random small fragments and then sequence them.

Question 13

Next-generation sequencing (NGS), also known as high-throughput sequencing, includes a number of different modern sequencing technologies:

Answer 13

• Illumina (Solexa) sequencing
• Roche 454 sequencing
• Ion torrent: Proton / PGM sequencing
• SOLiD sequencing

Question 14

Advantages of NGS over Sanger

Answer 14

Speed:
- In NGS, chain generation and signal detection are coupled, in Sanger they are two separate processes
- NGS allows for massive parallel sequencing, whereas in Sanger sequencing, one sequence is generated at a time (of around 1kB). In Illumina, 300Gb can be sequenced on the same chip.

Cost:
- Due to increased speed, lower reagent costs, the cost of NGS sequencing is much lower for large sequencing projects. Human genome sequencing might cost 10x less with NGS (less than 1,000€ in 2020)

Accuracy:
-individual reads are less accurate, but due to the parallel sequencing of many (overlapping) fragments, very reliable sequences are generated with NGS

Question 15

What is Recombinant DNA

Answer 15

• Recombinant DNA is DNA that has been created artificially.
• DNA from two or more sources is joined together into a single recombinant molecule.
• Made by ligating an insert into a vector.

Question 16

What is a (cloning) vector?

Answer 16

• A vector is a DNA molecule that acts as a vehicle to carry our insert into cells.
• It facilitates replication of the recombinant DNA construct in the cells.
• it can facilitate expression of the recombinant DNA construct in cells (only expression vectors).
• DNA fragments/inserts are also much more stable after they have been inserted into a vector (storage).

Question 17

Different types of cloning vectors

Answer 17

Plasmid vector- pBR322, pUC18/19
Cosmic vetor- pJB8
Phagemid vector- pEMBL8
Bacteriophage vector- M13
Artificial chromosome- PAC, BAC, YAC

Question 18

Essential properties of a ‘Vector’:

Answer 18

• you have to be able to isolate it from and re-introduce it into host cells.
• it has to be able to replicate independently in host cells
• it has to have at least one unique restriction enzyme site, so you can introduce your DNA

Question 19

Desirable properties of a ‘Vector’:

Answer 19

• Multiple cloning site (MCS), containing several unique restriction sites
• Selectable marker for presence of vector (antibiotic resistance genes)
• Selectable (selection) marker for presence of insert (e.g. lacZ gene)
• high-copy numbers can be produced in the host cells

Question 20

Main criteria for choosing a vector

Answer 20

• capacity for DNA insert (kb size)
• vector efficiency
• For expression vectors: must be suited for cell system, inclusion of tag sequences to facilitate purification and detection of the expressed protein

Question 21

Uptake of genetic material into E.coli can occur in 3 ways:

Answer 21

1. Transformation
2. Conjugation
3. Transduction

Question 22

Transformation

Answer 22

• Uptake of ‘naked’ (plasmid) DNA
• Not very efficient, only very few bacteria actually take up the plasmid (1:1000)
• Bacteria need to be ‘competent’

Question 23

Conjunction

Answer 23

• Transfer occurs between two cells in direct contact (pili)
• F-plasmid and BACs, very low copy number (1 copy/cell)

Question 24

Transduction

Answer 24

• Injection by bacteriophage infection
• Very efficient, close to 100% efficiency (every cell infected)

Question 25

Bacterial plasmid vectors

Answer 25

Bacterial plasmids are extrachromosomal, circular genetic material with their own origin of replication

Question 26

Where are plasmid vectors for recombinant DNA technology are derived from?

Answer 26

They are derived from natural R-plasmids, but have been modified, multiple cloning site, no oriT (for transfer between cells).

Question 27

What are the advantages and limitations of Bacterial plasmid vectors

Answer 27

Advantages:
- very easy to handle and to manipulate
- Can also be engineered for use in eukaryotic cells
Limitations:
- size of insert limited to around 10kB
- Uptake by transformation very inefficient

Question 28

Properties of Bacterial Artifical Chromosomes (BACs)
Based on E. coli F-plasmid

Answer 28

– Naturally occurring E.coli plasmid, “Fertility” factor
– E.coli cells carrying this plasmid are termed ‘male’, ‘female’ cells lack the plasmid
– 95 kB, big for a plasmid
– Conjugative transfer between E.coli cells.
– Extrachromosomal or integrated into host genome

Question 29

What Is BAC used for?

Answer 29

Used in large genome projects, such as the Human Genome Project

Question 30

What are vectors that can take up large fragments are mostly needed for?

Answer 30

genomic library generation

Question 31

what is a Gene libraries

Answer 31

A gene library is a collection of different DNA fragments from an organism, each of which has been cloned into a vector for ease of purification, storage, and analysis.

Question 32

what are the two types of gene libraries?

Answer 32

genomic libraries and cDNA libraries.

Question 33

what does the number of clones in a representative library depend on?

Answer 33

the genome size (genomic libraries) or the number of the expressed genes in a tissue or organism (cDNA library).

Question 34

what does a ‘gene library’ allow you to do?

Answer 34

A ‘gene library’ allows you to split up a complex mixture of DNA fragments into individual plasmids and bacterial clones.
Only one plasmid (i.e. one gene fragment) per E.coli clone.

Question 35

Genomic libraries: An example

Answer 35

The human genome:
3 billion base pairs (3 x 10E9 bps) on 24 chromosomes

The RPCI-11 Human Male BAC Library
-generated from human male genomic DNA (from blood cells)
-partially digested with EcoR1
- Size selected inserts cloned into pBAC vector (average insert size 178 kb)
-Total number of clones 543,797

Question 36

What does partial restriction enzyme digestion allows cloning of overlapping fragments

Answer 36

• partial digestion with a frequent-cutter allows production of overlapping fragments, since not every site is cut
• overlapping fragments ensures that all sequences in the genome are cloned
• overlapping fragments allows larger physical maps to be constructed as contiguous chromosomal regions (contigs) are put together from the sequence
  data

Question 37

Genomic libraries

Answer 37

• For genomic libraries, the entire genome is fragmented and cloned.
• Genomic library contains also introns and non- transcribed DNA.
• One clone does not necessarily contain a whole gene.
• One gene is likely to be split up between multiple clones.
• All genes should be represented equally.

Question 38

cDNA libraries

Answer 38

• cDNA libraries reflect the mRNA content of cells.
• Gene B is transcribed at a higher rate: more mRNA=more clones represented in cDNA library
• cDNA library derived from different tissues have different representation of genes!
• No introns! One clone contains the uninterrupted ORF of one gene.