L5 Libraries

Question 1

Genome mapping

Answer 1

Represents the distance between 2 DNA elements e.g Lysis gene and hair gene on chromosome 3 is X-distance apart.

Based on recombination freq

Genes used as markers

Limited degree of resolution

Question 2

Physical mapping

Answer 2

Maps of genetic markers obtained by direct analysis of DNA sequence.

Restriction mapping

Generated genomic libraries

Preliminary step to genome sequence.

Relating a gene to chromosomal positioning by markers

Question 3

Restriction mapping

Answer 3

Analysis of fragments after being cut by restriction enzymes.

Allows to locate where the restriction sites are located

Only applicable to small DNA molecules.

Cannot map entire genome

Question 4

Fluorescence in-situ Hybridisation (FISH)

Answer 4

Allows the position of a fluor marker on a chromosome to be visualized.

Used to map chromosomal location of particular genes

Method:

1) Denature during metaphase
2) Add fluorescent probe
3) Fluorescence is emitted to a part of chromosome

Modification:
Mechanically stretched chromosomes - Markers are 200-300 kb apart
Fiber-FISH - 10kb apart

Question 5

Genomic libraries

Answer 5

High mol. weight DNA > Fragmentation > less than 50kb > obtain genome sequence

The fragments are then cloned.

Question 6

DNA Fragmentation

• Mechanical shearing
• Restriction Enzyme digestion

Answer 6

Shearing:

• Sonication of DNA
• Cannot control size of desire and inefficient

Restriction:

• Digestion of genomic DNA followed by cloning in vector
• Sizes are controlled by the exposure time of DNA to enzymes

Question 7

Cloning strategies for genomic libraries

• Plasmid
• Phage
• Cosmid/BAC

Answer 7

Plasmid:

• Fragment size is limited
• Low relative transformation efficiency

Phage:

• Insert capacity limited (size)
• Efficient

Cosmid/BAC:

• Higher insert capacity
• Easier to handle

Question 8

Number of clones needed (maths)

Answer 8

f = no. of clones

f = Genome size/Insert size

f is an under-estimate

Bad:

• sampling error
• hard to clone repetitive sequences

Question 9

cDNA library

Answer 9

Only 1.5% of genome contributes to proteins.

Eukaryotic genes contain exons (coding) and introns (non coding)

Question 10

mRNA copied into cDNA for cloning

Answer 10

mRNA cannot be cloned into a vector. Hence must be copied into cDNA

Process: Reverse transcription via reverse transcriptase

Synthesis in 5’ to 3’
Requires a primer and template

Question 11

mRNA extraction by Oligo-dT affinity chromotography and cDNA synthesis

Answer 11

The polyA tail is a unique feature to mRNA, allows isolation from tRNA and rRNA

1) Use Oligo-dT to bind mRNA
2) Wash away rRNA and tRNA
3) Reverse transcriptase adds basses
4) Remove RNA by alkaline or RNAse
Add polyG tail
5) Hybridize PolyG tail of cDNA with oligo-dC primer
6) New complimentary strand

Question 12

Cloning cDNA

Answer 12

Restriction site linker

1) T4 DNA ligase
- ligates cDNA (blunt) fragment to “linkers”
2) Linker is cut by EcoRI to get sticky ends
3) Hybridized with vector

Homopolymeric tailing

1) Add terminal transferase + dCTP, adds a polyC tail to cDNA
2) Add terminal transferase + dGTP to digested vector, adds a complimentary polyG tail.
3) T4 DNA ligase to hybridize

Question 13

Storing and propagating libraries

Answer 13

Once the primary library is contained, it can be diluted in suitable solution and plated again to recolonize.

Phage libraries stored at 4 degrees, months

Cosmids, 20% glycerol sln at -80 degrees, moths/years

Question 14

Sequencing whole genomes

• Shotgun
• Hierarchy shotgun

Answer 14

Shotgun: searching for overlaps between fragmented sequences

Hierarchy: Contig assembly is used. Identifies by using PCR and hybridisation

Question 15

Metagenomics

Answer 15

Direct analysis of DNA from environmental sample.