Lecture 28 - Genetic Mapping and SNPs

What are 5 different techniques in biotech and genetics?

1. PCR - amplification

2. Genomics

3. Genetic maps

4. Positional cloning

5. Site directed mutagenesis

Whar are 3 types of chromosomal maps?

1. Genetic

2. Cytological

3. Physical

Genetic maps

Based on recombination frequencies

Cytological maps

Based on banding patterns of stained chromosomes

Physical maps

Based on molecular distances between regions on the chromosomes

What are 4 reasons to map chromosomes?

1. Positional markers used for genome sequencing

2. Useful for pinpointing disease causing genes

3. Distinguishes between individuals (forensics)

4. Provides insight into the architecture of the genome and hotspots for activity

What is required for genetic mapping?

Allelic variation including the frequency of recombination between 2 linked genes AND frequency of recombination of 1%

Polymorphic markers

Markers with high frequency of allelic variation

What does RFLP stand for?

Restriction fragment length polymorphism

What do mutations in restriction enzyme cut sites lead to?

Allelic differences (variation in fragment length)

Where can positional markers be linked to?

Physical and genetic maps of chromosomes. AND other polymorphic markers such as gene for a disease

Microsatellites

A polymorphic marker that is made up of a short segment of DNA

How are markers located on a cytological map?

Probes (ex. sequence around an RFLP) are hybridized to a condensed chromosome. Both probe and chromosome are denatured for complementary base pairing

Describe linking different types of markers

Recombination occurs less frequently near centromeres and telomeres. DNA is not evenly packed in different regions of a chromosome

What is positional cloning used for

Identifies disease causing genes

How does positional cloning work?

Polymorphic markers are used to locate the approximate region of disease gene. DNA sequence daround polymorphic marker to find disease gene

How is Huntington’s disease (a dominant trait) identified?

The RFLP marker that is closely associated with the disease is identified

What are the steps to positional cloning?

1. Nearest genetic marker (RFLP or microsatellite) is identified through pedigree analysis

2. Marker used to identify a genomic clone from a genomic library of an individual with a specific disease

3. Clone is screened for the gene. Sequencing is used to identify mutant genes and the wild type homologue is sometimes tested for its ability to restore mutant to WT (same as gene therapy)

What are 4 methods to exploring the genome beyond DNA sequencing

1. RNA sequencing

2. Epigenetics

3. Single Nucleotide Polymorphisms (SNPs)

4. Green fluorescent protein fusions

SNP chips

They identify specific gene variants in individuals

How do SNP chips work?

1. SNPs and flanking DNA are applied to the chip

2. SNPs are amplified (through PCR) from different individuals and hybridized with the chip

3. Chip is washed and only the exact match with the chip SNP bind will NOT wash away

SNPs and personalized medicine

Some haplotypes are more sensitive to a drug than other haplotypes. Drug dosage can be optimized based on the haplotype to maximize effectiveness

Genome wide association studies

Aims to identify genes with observable traits without directly hunting for genes. Studies determine whether specific SNPs are more commonly found in group with the disease than the control group

GWAS requirements

1. Requires a group of people with disease and one without it (control group)

2. Uses a large number of polymorphic markers such as SNPs

Problems with GWAS

1. Studies are expected to detect disease causing genes if common diseases are associated with common causing mutations

2. Association between marker and disease are often weak

Green fluorescent protein fusions

1. GFP is placed next to gene of interest (fusion protein)

2. Shows expression of proteins in living cells and appears fluorescent green when exposed to UV or blue light