Mapping the Genome Flashcards
Explain the principles of linkage analyses Describe the difficulties of linkage analyses in humans and how they're addressed Demonstrate an understanding of key physical mapping techniques Explain how physical & genetic mapping are used together to identify functional genome sequences Describe the principles & uses of population based gene mapping methods
Describe what is meant by a genome map
A diagram of the chromosome which shows the positions of key features such as genes and control regions. Essential in investigating how the genome works and identifying which regions of the genome are medically important.
Describe a cytogenetic map
AKA karyotype. Under Giemsa staining it shows light and dark bands in the chromosome. This type of map contains the least detail.
Describe what is meant by a genetic map
Shows relative positions of genes (or sequence polymorphisms) within the genome. These type of maps are generated by linkage analysis - measures the frequencies with which two phenotypes are coinherited.
How are genetic distances measured and what do they mean?
If two alleles are close together, it shows that they have a higher probability of being co-inherited. Genetic distances are measured in centimorgans (cM). Linkage analysis can measure the genetic distance between gene & sequence polymorphisms, but it can’t map genes that underly multigenic phenotype. Logarithm of odds (LOD) scores can be used to quantify linkage - provides a statistical estimate of whether 2 genes are likely to be co inherited.
What is meant by a physical map?
A map of the DNA generated by direct examination, often using restriction enzymes and sequencing. Ultimately generates complete sequences & measures distance in base pairs. Large genomes may need to be split up.
How are physical maps annotated?
Use techniques to determine the important functional elements such as genes. Annotated using info genetic mapping, comparative genomics, reverse genetics, CHIP-seq, Genome Wide Association Studies. GWAS locates genomic regions determining complex phenotypes.
Describe linkage analyses and explain what it shows us
It relies on crossing over - if there was no crossing over, alleles on the same chromosome would always be co inherited. Genes that are close together have a low chance of being separated = small recombinant phenotype frequency.
How do we calculate recombination frequency and what does it show us?
Recombinant offspring/Total offspring %. 1% RF is a centiMorgan. A 50% recombination frequency means that the genes are far apart or on different chromosomes, it is the maximum RF value.
How does the genome arrangement of a human make linkage analyses difficult? (no. of autosomes) How can we overcome this?
For linkage analyses, the genes need ot be on the same autosome and humans have 22 so the chances are lower. We detect if two genes are on the same autosome by fusing human and rodent cells to create a hybrid cell, and then growing it into cells containing different subsets of human chromosomes. We can tell which chromosomes they contain by G banding, and then we can tell which proteins are expressed by PCR or Western and compare the data.
What limits do human pedigrees have and how do we overcome these?
We cannot selectively breed humans so the parental genotype is hard to establish. Also, human pedigrees are often small, especially when inflicted with a genetic disease - limiting the number of meioses that can be analysed (increased progeny = increase map resolution). Because of this, statistical linkage analysis (mathematical model) is used. Uses a parametric LOD score which is a log ratio of linkage likelihood/no linkage likelihood.
Establishing linkage requires both alleles of the gene to be present in the pedigree - these genes are informative. Most genetic disorders are rare so most families don’t have informative genes.
DNA cloning -> using DNA markers more. DNA markers are any polymorphic (variable within a population) sequence. Most common is SNP - occurs 1/300 bp. If a SNP makes/destroys a restriction site then it changes the DNA fragment - restriction fragment length polymorphism (RFLP).
How are DNA markers used in linkage analyses?
Micro and mini-satellites (tandemly repeated sequences) are also highly polymorphic. Polymorphisms at these loci are called variable number of tandem repeats (VNTRs) or simple sequence length polymorphisms (SSLPs) and are useful DNA markers. PCR/Electrophoresis/Sequencing can find the markers in pedigrees and linkage analyses can be used to see how they co-segregate with disease genes.
Explain the process of Shotgun sequencing
The genome is broken up into 50-200kb overlapping fragments. The larger fragments are cloned into bacterial artificial chromosome (plasmid). The ends of the fragments are sequenced and compared, the overlaps allow the correct order to be determined, but repeated regions make it hard. The larger continuous fragments (contigs) are split up and sequenced.
What are the differences between functional and positional cloning?
Functional cloning is using the function to determine the location of the gene (identifying faulty proteins and finding genes from there). Positional cloning is when you don’t know the function of a gene, and you locate it by using linkage analyses.
Why is it that most variation amongst the population does not lead to disease?
This is because any disease causing variation would most likely be selected against.
