L10 Forward And Reverse Genetics

Two ways to study genes and their functions

一、The Forward Genetics Approach

Forward genetics is a phenotype-to-genotype approach

Connects gene functions to gene sequences
Forward genetics requires no knowledge of gene products
Genetic variation or mutations are the starting point
Mutations induced through mutagenesis

Mutagens most commonly used in plants:

Fast neutron / gamma rays – large deletions
EMS (ethyl methanesulfonate) – point mutations
T-DNA – single insertions, slow to generate
Transposons – more insertions, easy to generate

1. Complementation Test

Methods of Forward Genetics

(How can you find mutated genes that cause phenotypes?)

1. Walking – Map-based / positional cloning

(1) Introduction

Look for genetic regions associated with mutations, or exclude chromosomal segments that contain no mutations

Need for a mapping population
DNA markers
Genomic DNA clones (large size)

Map-based Cloning

Map-based cloning is an exercise of converting a genetic map to a physical map

Linkage analysis is the basis of map-based gene cloning

Recombination is the basis of linkage analysis.

Use genetics to map trait to an interval to find tightly linked markers.

利用遗传学将性状映射到一个区间，以找到紧密相连的标记。

Use marker to identify BAC clone.

BAC(Bacterial Artificial Chromosome，细菌人工染色体）

Use other end of BAC to make a new marker, check for recombinants. Find next clone, repeat until gene is flanked by clones. （找到下一个克隆体，重复这个步骤，直到克隆体包围了基因。）Transform clones to complement mutant. Sequence the gene and determine if the function is known. Sequence clone.

(2) Steps

Making a mapping population and selection of markers close to the mutant locus
Selection of large clones covering the mutant locus (Optional when genome sequence is available)
High-resolution mapping of the mutant locus
Search for a Candidate Gene and Mutations
Confirmation of cloning by transgenic complementation

Recombination Frequency Is Proportional to Genetic Distance

1% RF = 1 map unit, a centimorgan (cM)

The closer the distance between two loci, the less frequent is the recombination.

两个基因座之间的距离越近，重组的频率就越小。

High resolution mapping is the process of finding markers that demonstrate fewer or no recombination events.

2. Tagging (transposon or even T-DNA tagging)

Look for genes associated with insertions

Need for insertion sequences

Working Mechanism

The closest promoter is activated by T-DNA borne enhancer element

EN is not orientation-dependent

Activation can range over 12 kb

‘One EN-One Gene’ activation

Dominant, Gain-of-Function phenotype

Phenotype can be easily copied in other plants

3. Direct sequencing

Need a good reference genome

Mutant must be in the same reference genotype

一、Reverse Genetics

Reverse genetics is a genotype-to-phenotype approach that connects gene sequences to gene functions.

Gene sequences are the starting points of reverse genetics (e.g. knowledge of your candidate gene is required)

Characterics of Reverse and Forward Genetics

The Process of Reverse Genetics

Useful Candidate Data

Expression Pattern – microarray datasets, EST projects, sequence tag libraries, northern analysis, in situ hybridization
Homology – members of an interesting gene family, similarity to genes that may perform a similar function in other organisms
Domain information, such as protein-protein interactions – yeast two-hybrid screen, protein complex isolation/mass spec