Lecture 21

Question 1

What is a Genomic Library?

Answer 1

A collection of all the genomic DNA fragments of a given species that have been taken from one organism and inserted into a vector for cloning

Question 2

What is the aim of a Genomic Library?

Answer 2

Gain a panel of bacteria containing individual clones that represent all of the DNA in an organism’s genome

Question 3

What genomic DNA can be used to make a Gene Lib?

Answer 3

1. Eukaryotes
2. Prokaryotes
3. Viral

Question 4

What factor determines which vector to use?

Answer 4

The size of the genome

Question 5

What are the two possible vectors and describe their relative size

Answer 5

1. Plasmid = 20kb each

2. Bacterial Artificial Chromosome Vector = 150kb each

Question 6

Describe the shared features of a BAC and the F- Plasmid

Answer 6

1. Low copy number
2. Contain genes to stabilise the plasmid
3. Antibiotic resistance marker
4. Can carry large DNA inserts

Question 7

Describe how bacterial genomes are put into an expression library

Answer 7

Prokaryotic DNA -> transcribed into mRNA -> translated into proteins

Question 8

How is eukaryote RNA considered pre/post intron splicing?

Answer 8

• Pre = primary

- Post = mature

Question 9

What is the solution around introns in eukaryotic genomes?

Answer 9

Clone the mRNA and make cDNA libraries

Question 10

What is cDNA?

Answer 10

Complementary DNA is a DNA copy synthesised from mRNA using a viral genome (reverse transcriptase)

Question 11

What can Reverse Transcriptase do?

Answer 11

Can turn RNA -> DNA

Question 12

What is the results of using Reverse Transcriptase?

Answer 12

DNA sequences will lack introns

->therefore can be translated into bacterial proteins

Question 13

What is a cDNA library and what does it reflect?

Answer 13

• A collection of all expressed RNA’s

- Reflects what tissue type we use under the circumstances at a particular time

Question 14

What are ways to make cDNA libraries?

Answer 14

1. Can order cDNA clones

2. Can synthesise cDNA

Question 15

Describe the 7 steps to Sanger Sequencing

Answer 15

1. DNA to be sequenced will align
2. Heat is applied to separate the strands
3. Anneal a primer
4. Extend strand with 4 dideoxynucleotides (each with a different fluorescent dye)
5. Polymerase extends primer and gets range of extended products ended by dye labeled terminators
6. Separated on a capillary gel
7. Bands detected with a laser

Question 16

Describe the steps of ShotGun Sequenceing

Answer 16

1. Cut the genome
2. Clone it
3. Use vector DNA as primers
4. Sequence the vectors and generate many small fragments
5. Join the sequences which creates a contig (these are overlapped to produce a full sequence)

Question 17

How much of the genome does ShotGun sequencing sequence?

Answer 17

The whole genome

Question 18

What is the output of ShotGun sequencing?

Answer 18

A random collection of short sequences, some overlapping (contigs)

Question 19

Describe DNA cloning steps in making the COVID Vaccine

Answer 19

1. Plasmid DNA containing SARS-Cov-2 spike protein inserted into bacteria. Bacteria will replicate for 2 weeks producing lots of plasmids
2. Plasmid DNA is purified
3. Plasmid DNA is linearised using Restriction Enzymes
4. Spike protein DNA is transcribed into mRNA
5. Spike protein mRNA is bound with lipid particles for protection