Bioinformatics 3: Genome organisation and evolution

Question 1

What are transcriptomes? Proteomes?

Answer 1

Transcriptomes -> what genes are active in time + space

Proteomes -> what (protein coding) genes are doing

Question 2

Polymorphic DNA variants? What characteristics must they possess?

Answer 2

Restriction fragment length polymorphisms (RFLPs)

Simple sequence length polymorphisms (SSLPs) - mini and microsatellites

Single nucleotide polymorphisms (SNPs)

Must be:

• Common in the genome
• Show mendelian inheritance
• Have low mutation rates
• Easily typed by molecular biological methods

Question 3

Method of typing Restriction Fragment Length Polymorphisms?

Answer 3

PCR and restriction digestion (RFLPs)

-> if digested band is present, site is present, if not, site is absent

Question 4

Method of typing Simple sequence length Polymorphisms (SSLPs)
and/or
Simple tandem repeats (STRs) ?

Answer 4

PCR amplification and sizing (SSLP/STRs)

-> amp + size each individual allele creates individual DNA fingerprint

Question 5

Method of typing single nucleotide polymorphisms (SNPs)?

Answer 5

Solution hybridisation (SNPs)

-> Quenching compound and fluorescent compound bind to DNA, if SNP is present, strentching occurs -> quenching compound moves away and you get fluorescence

Question 6

3 techniques used in physical genetic mapping?

Answer 6

Restriction Mapping

Sequence Tagged Site (STS) Mapping

Fluorescence in situ Hybridisation (FISH) Mapping

Question 7

Describe the basis of restriction mapping

Answer 7

Restriction enzymes (REs) digest DNA fragment, either:

• Complete -> REs digest fragment into multiple fragments
• Partial -> REs, often in sub-optimal conditions incompletely digest fragments, some larger, undigested fragments left, useful for solving existing maps

Question 8

Advantages/Disadvantages of restriction mapping?

Answer 8

Most REs cut too often at genomic level -> enzymes with rare recognition more useful

• Limited by ability to accurately size resultant fragments using gel electrophoresis

Question 9

What is BAC/PAC fingerprinting?

Answer 9

RFLP mapping of large insert cloning vectors ->

High throughput fingerprinting gel using BAC/PAC

Question 10

Describe the principles of Sequence tagged Sites (STS) mapping

Answer 10

STS - unique sequences detectable by hybridisation / PCR

Any randomly fragmented DNA collection can be used in STS mapping

Question 11

describe process of STS Mapping

Answer 11

X-ray irradiation of cells -> produce chromosome fragments

Fusion of irradiated cells with hamster cells

Cell lines cloned and selected to contain fragments from a few or one human chromosomes

STSs must be specific to human DNA

Question 12

Describe the basis of Fluorescent in situ Hybridisation mapping

Answer 12

FISH enables physical localisation of fluorescently labelled DNA sequences to metaphase chromosomes

Question 13

Problems with FISH mapping?

Answer 13

Repetitive DNA content of probes (blocking)

Resolution (Metaphase chromosomes ~1Mb)
(some DNA prep methods can solve this e.g. centrifugal stretching)

High labour, low throughput

Question 14

Order of gene mapping processes in Cystic Fibrosis case study? (before human genome was sequenced)

Answer 14

Linkage Mapping
STS Mapping
Refinement (of linkage)
Cloning - many potential genes identified

Candidate genes checked for presence in cDNA libraries - one transcript found

Gene identified as encoding a transmembrane domain containing protein, confirmed with knockout mice

Question 15

perhaps more on prokaryotes

Answer 15

d