Lecture 21 Flashcards
What is a Genomic Library?
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
What is the aim of a Genomic Library?
Gain a panel of bacteria containing individual clones that represent all of the DNA in an organism’s genome
What genomic DNA can be used to make a Gene Lib?
- Eukaryotes
- Prokaryotes
- Viral
What factor determines which vector to use?
The size of the genome
What are the two possible vectors and describe their relative size
- Plasmid = 20kb each
2. Bacterial Artificial Chromosome Vector = 150kb each
Describe the shared features of a BAC and the F- Plasmid
- Low copy number
- Contain genes to stabilise the plasmid
- Antibiotic resistance marker
- Can carry large DNA inserts
Describe how bacterial genomes are put into an expression library
Prokaryotic DNA -> transcribed into mRNA -> translated into proteins
How is eukaryote RNA considered pre/post intron splicing?
- Pre = primary
- Post = mature
What is the solution around introns in eukaryotic genomes?
Clone the mRNA and make cDNA libraries
What is cDNA?
Complementary DNA is a DNA copy synthesised from mRNA using a viral genome (reverse transcriptase)
What can Reverse Transcriptase do?
Can turn RNA -> DNA
What is the results of using Reverse Transcriptase?
DNA sequences will lack introns
->therefore can be translated into bacterial proteins
What is a cDNA library and what does it reflect?
- A collection of all expressed RNA’s
- Reflects what tissue type we use under the circumstances at a particular time
What are ways to make cDNA libraries?
- Can order cDNA clones
2. Can synthesise cDNA
Describe the 7 steps to Sanger Sequencing
- DNA to be sequenced will align
- Heat is applied to separate the strands
- Anneal a primer
- Extend strand with 4 dideoxynucleotides (each with a different fluorescent dye)
- Polymerase extends primer and gets range of extended products ended by dye labeled terminators
- Separated on a capillary gel
- Bands detected with a laser
Describe the steps of ShotGun Sequenceing
- Cut the genome
- Clone it
- Use vector DNA as primers
- Sequence the vectors and generate many small fragments
- Join the sequences which creates a contig (these are overlapped to produce a full sequence)
How much of the genome does ShotGun sequencing sequence?
The whole genome
What is the output of ShotGun sequencing?
A random collection of short sequences, some overlapping (contigs)
Describe DNA cloning steps in making the COVID Vaccine
- Plasmid DNA containing SARS-Cov-2 spike protein inserted into bacteria. Bacteria will replicate for 2 weeks producing lots of plasmids
- Plasmid DNA is purified
- Plasmid DNA is linearised using Restriction Enzymes
- Spike protein DNA is transcribed into mRNA
- Spike protein mRNA is bound with lipid particles for protection
