GEN 5: Mapping the Genome Flashcards
1
Q
Observe the learning outcomes of this session
A
2
Q
What is a genome map?
A
- a genome map is a diagram of the chromosome(s) showing the relative positions of key features, such as genes and control regions
- they are essential for investigating how the genome works and identifying disease genes and other regions of medical importance
3
Q
What are cytogenetic maps/karyotypes?
A
- cytogenetic maps, or karyotypes, represent the visual appearance of chromosomes when stained and examined under the microscope
- they have the lowest resolution compared to the two main kinds of maps: genetic maps and physical maps
4
Q
What are genetic maps?
A
- genetic maps show the relative positions of genes (or DNA sequence polymorphisms within the genome
- they are generated by linkage analysis
- linkage analyses measure the frequencies with which two phenotypes are co-inherited
- this proves an estimate of how close their underlying genes are to each other on a chromosome
- the resulting genetic distances are measured in centimorgans (cM)
- linkage analysis can also be used to measure the genetic distance between a gene and a DNA sequence polymorphism, but it cannot be used for mapping the genes that underlie a complex (multigenic) phenotype
5
Q
What are physical maps?
A
- physical mapping involves direct examination of DNA by a range of techniques including:
- restriction enzyme mapping
- DNA sequencing
- it generates the complete genome sequence and measures distances in base-pairs
- high throughput sequencing can generate complete sequences of small genomes, and larger genomes may need to be sequenced in segments and pieced together by referring to a genetic map
6
Q
How are physical maps annotated?
A
- An informative DNA sequence map (such as those seen in genome browser) requires sequence annotation to show the positions of genes and other functional elements.
- This is an ongoing process that combines information from several approaches including genetic mapping, comparative genomics (GEN4), reverse genetics (GEN9), CHIP-seq (GEN7) and Genome-Wide Association Studies (GWAS).
- GWAS are used to locate the genomic regions that determine complex phenotypes; they were briefly mentioned in GEN4 and will be described further here.
7
Q
How was linkage analysis developed?
A
- it was developed from a finding that some traits broke Mendel’s law of Individual Assortment:
- ‘The alleles of two (or more) different genes get into gametes independently of one another’
- this occurs if the genes are located on different chromosomes
8
Q
What are linked genes?
A
- some genes ‘break’ the law of independent assortment
- this occurs if genes are located on the same chromosome
- they are very close they will always be inherited together
9
Q
What are partially-linked genes?
A
- some genes behave in an intermediate manner
- where they are sometimes inherited together and sometimes they segregate to form recombinant phenotypes
- this is dependent on crossing over
10
Q
How do partially-linked genes occur?
A
- this occurs due to the crossing over that occurs during meiosis
- Without meiotic recombination all alleles on the same chromosome would always be co-inherited
- However, crossing over has the potential to separate two alleles.
- During meiosis each chromosome will undergo between one and four cross-over events.
- The cross over is essentially random, although some regions are slightly more prone to crossing over.
- Therefore for genes widely spaced on the chromosome they are likely to be separated by a cross over event during every meiosis.
- Conversely the closer two genes are to each other on a chromosome, the smaller the chance that they will be separated by a cross-over during meiosis, and so the smaller the frequency of recombinant phenotypes.
11
Q
Describe an example of linkage in Drosophila
A
- two genes on the same chromosome determining body colour (grey or black) and wing development (normal or vestigial)
- the linkage was studied by using mating between different genotypes
- alleles B (grey bodies) and Vg (normal wings) are dominant over alleles b (black bodies) and vg (vestigial wings)
- info about the parental gametes are in first card
- if the genes were very close together, then the chance of a cross-over between them would be minimal and very few, or no, recombinant progeny would be expected
- For increasingly large distances between the two genes, however, recombinant progeny would be expected in increasing numbers until all recombinant and parental phenotypes occur at similar frequencies (no linkage). The actual numbers of the different phenotypes that were observed is shown at the bottom of the following summary image
12
Q
What is the recombination frequency?
How do you calculate it?
A
- the proportion of progeny with recombinant phenotypes is called the recombination frequency
- recombination between loci can be calculated as:
RF = (recombinant offspring / total offspring) x 100%
13
Q
What type of crossover allows recombination events to be seen in the phenotype of the offspring?
A
- by crossing a parent who is heterozygous for the two genes with a parent who is homozygous for the recessive allele at the two genes
- this means that recombination events can be seen in the phenotype of the offspring
14
Q
What does an RF of 1% represent?
A
- an RF of 1% is defined as one centimorgan (cM)
- it is estimated that in humans one cM is roughly 1200 kb
15
Q
The Recombination Frequency (RF) for two genes is inversely proportional to the distance between them.
True or False?
A
- False
16
Q
An RF value of 50% for two genes indicates that they are either far apart on the same chromosome or on different chromosomes
True or False?
A
- true
18
Q
Why is assigning genes to autosomes a difficulty for human linkage analysis?
What is the solution?
A
- genetic linkage between two genes requires them to be on the same chromosome
- the chance that two unmapped autosomal genes are on the same autosome is reasonably high in flies (3 autosomes), but much lower in humans (22 autosomes)
- a way of knowing if the two genes are on the same autosome is needed
Solution:
- before we had access to the whole genome sequences, the solution was to use somatic cell hybrids
- these are generated by fusing human and rodent cells
- during subsequent rounds of cell division, human chromosomes are lost, by an unknown mechanism
- the hybrid cells formed have the whole rodent genome but just a subset of human chromosomes
- the human chromosomes present can be characterised using G-banding
- it is then possible to search for a gene of interest in the hybrid cell lines by performing western blot or PCR
- in this way, a gene can be assigned to a specific autosome
19
Q
Why are human pedigrees being limiting a difficulty for human linkage analysis?
What is the solution?
A
- knowing and being able to select the genotype of the parents facilitates linkage analysis
- establishing parental genotypes is often difficult in human families and breeding partners clearly cannot be chosen by the investigator
- furthermore, human pedigrees, especially those affected by genetic disease, are often small, limiting the number of meioses that can be analysed
- this is problematic because the resolution of genetic maps increases with the number of progeny scored
solution:
- statistical linkage methods were developed
- these use mathematical models to establish how likely it is that two phenotypes are truly linked and do not just happen to segregate by chance
- The classic statistical approach to linkage analysis is to calculate a ‘parametric LOD score’
- LOD stands for ‘logarithm of odds’
- It is the log-ratio of the likelihood linkage over the likelihood of no linkage.
- look at the table to explain how scores relate to a probability that two genes are linked rather than randomly co-segregating
- a LOD score greater than 3.0 indicates 1 in 1000 odds the linkage observed occurred by chance and by convention is considered to be evidence of linkage
- LOD scores can also be negative and by convention a LOD score less than ~2.0 is considered evidence to positively exclude linkage
20
Q
Why is it that informative genetic loci being rare a difficulty for human linkage analysis?
What is the solution?
A
- to establish linkage between two genes in a human family, each allele must be present in at least two distinguishable forms in the family pedgree
- e.g. causing a disease or a phenotype
- such genes are said to be informativve
- most genes studied at the time however, were for rare genetic disorders and the chances of finding families in which linkage between two such disease genes could be tested was very low
solution:
- with the development of DNA cloning, the use of DNA markers for linkage mapping became widespread
- this could be any piece of genomic DNA whose sequence is polymorphic
- they became informative DNA genetic markers, because their inheritance through the pedigree could be followed
- single nucleotide polymorphisms (SNPs) are the most informative genetic markers
- this is a particular nucleotide position in the genome with a variant in at least 1% of the populatioin
- on average, a SNP occurs every 300bp in the human genome sequence, so SNPs are a rich source of variation
- Sometimes a SNP will create or destroy a recognition site for a particular restriction endonuclease, leading to a change in the DNA fragments it generated.
- These polymorphism are called a restriction fragment length polymorphisms (RFLPs)
- tandemly repeated sequences, such as micro- and mini-satellites, are also highly polymorphic
- Polymorphisms at such loci are called variable number of tandem repeats (VNTRs), or simple sequence length polymorphisms (SSLPs) loci, and are extremely useful as DNA markers.
- using methods such as PCR and gel electrophoresis, or DNA sequencing, polymorphic DNA markers can be easily identified in family pedigrees.
- Linkage analysis can then be used to see how these markers co-segregate with a disease gene.
- This approach, as used with a VNTR DNA marker, is illustrated in the following image.
21
Q
Fill in the gaps
A
23
Q
Choose the most appropriate statement
A
B
25
Q
Describe the hierarchical shotgun approach used when sequencing the human genome
A
- this was a way of improving the reliability of the data acquired across repetitive DNA regions
- the genome was first fragmented to large pieces of 50-200 kb in length
- the larger genomic fragments were cloned into plasmids called a BAC (bacterial artificial chromosome) library
- the ends of these DNA fragments are sequenced and compared
- by identifying sequence overlaps the fragments can then be arranged in the correct order
- once this initial map has been assembled, a set of continuous fragments (contigs) can be fragmented into much smaller pieces and sequenced to generate a complete genomic sequence
- this hierarchical method is more time consuming but helps minimise the number of fragments sequenced and the computer power required to align and produce the full genomic sequence
26
Q
What is positional cloning?
A
- Any information about a disease gene and its predicted product has huge potential for designing new treatments and diagnoses.
- The combined use of genetic and physical maps allowed researchers to identify and clone important disease genes in a process called positional cloning.
- This approach was used to clone the genes for monogenic diseases such as cystic fibrosis and muscular dystrophy, when nothing was known about the altered protein or mRNA.
- In such cases, searches for the gene had to be made starting with only the genetic map position and looking for candidate genes in the equivalent position of the physical map.
- Because of the limited resolution of genetic maps, DNA regions as large as 20 Mb were screened for candidate genes, which were tested until one was found that was mutated in affected individuals.
- extra (untested) info in picture
27
Q
Observe the diagram of the differenced between functional and positional cloning
A
28
Q
Why is positional cloning not necessary in some diseases?
A
- For some genetic diseases, positional cloning was not necessary. In sickle cell disease, for example, it was known from other studies how the β-globin protein was affected and the β-globin mRNA was readily isolated.
37
Q
How do you interpret a Manhattan plot of GWAS results?
A
40
Q
Fill in the sentence with the correct words
A
