Ronald Flashcards
What are AFLPs?
Amplified fragment length polymorphisms. These are much more reproducible, stable and informative than RAPD’s.
What do RAPDs use and what is the problem with them?
RAPD’s use random 10mers (10 nucleotide primers) with a low stringency annealing PCR to amplify random fragments of the genome to generate fragments of varying size, that can be separated on a gel. As these are random they are anonymous markers and you can look for one which is polymorphic among the individuals of your cross, and that co segregates with your trait of interest. The problem with these is that they produce bands of varying intensity and are largely non-reproducible hence what might look like a correlation in one gel, may not exist if the experiment is repeated.
What differences are their in the AFLPs approach compared with RAPDs?
AFLP’s use a similar approach, but use a restriction enzyme digest and then use the sticky end overhangs created by the enzyme (s) to attach linkers/adaptors. These are of know sequence and you can reate complementary primers to them that are radioactively or fluorescently tagged. For larger genomes the number of bands produced will be immense so you can extend the primers 1, 2, 3 or however many nutcleotides you like, past the end of the adaptor sequence. These will be random nucleotides just to reduce the total number of fragments you have to deal with. For larger genomes it is safe to assume that a fair number of the fragments will, by chance, have sequences corresponding to the random sequence that you extended your primers by. Also you can use the GC content of the genome to be more precise, if you want to greatly reduce your number of fragments for a high GC content genome, extend the primers by A and T’s, or the reverse for a low GC genome or if you only want to slightly reduce the number of fragments you have to deal with.
Once you have your Gel what do you look for with RAPDs?
Once you have your gel, as for RAPD’s, you look for a polymorphic band that cosegregates with your trait of interest. Note: you are scoring for presence or abense of a band. From here you can cut out the band, amplify it using the same primers you used to generate the gel, and then sequence it (marker conversion) so the band is no longer anaonymous, as you know its sequence, and you can use it as a marker to probe a library.
Advantages lie in that you can use this technique for an organism and it allows you to map genes that may lie in regions of low marker density.
What are the stepgs in AFLP analysis?
Take two individuals that are isogenic (genetically identical, except for the trait of interest) and homozygous, this is so you start with maximum difference and minimum noise (similarities in results due to genetic similarities). For this type of cross you can use organisms such as drosophila where one sex is achiasmic. You cross these two to produce hybrid F1’s and then perform a testcross with the F1’s.
For the achiasmic sex, the F2 progeny they produce will not be the product of crossingover, so this allows you to assign linkage groups (markers that are on the same chromosome) amd see if any linkage group’s presence or absence can be correlate to presence or absence of your trait of interest.
For the chiasmic sex, you can work out the relative distance between markers using crossing over frequencies.
What is the BAC and what is it used for?
BAC: Bacterial artificial chromosome: This is a vector based on the bacterial F plasmid, that can hold 100-150kb of nucleic acid sequence for the purposes of generating a DNA library.
What do you do if you have a low marker density?
library.
For regions where marker density is low, you may want to initiate a chromosome walk and for this you need a BAC library (series of BAC clones all carrying different put partially overlapping DNA fragments that together, constitute the entire genome, or most of it, of an organism).
Once you have narrowed down the location of your gene of interest via linkage analysis, to between two knwon markers, you can use this markers to initiate a chrosmome walk. You probe a BAC library for the marker(s) to pick out clones containing that marker. You can determine the order of the clones through restriction enzyme digestion, and using the inserts at the ends of the region spanned by selected clones, sequence the ends of these clones and probe the library again.
Alternatively you can sequence the clones and design new primers to amplify them. You can them apply these primers to DNA of the original parents of the cross and see if the band is polymorphic. This is to aid in your construction of a genetic map to see if you can get any closer to your gene of interest. Eventually you want ot be at the point where your marker has a 0 recmbination distance from your gene of interest.
Your chrosomosome walks from the flanking markers will eventually reach a point wher the identified clones overlap and the region this spans defines a physical region in which your gene sits.
What is a genomic library?
Collection of partially overlapping fragments of genomic DNA, cloned into vectors.
What is a contig?
Series of overlapping clones that cover an entire chromosomal region
What is a physical map?
Series of contigs that span the entire genome.
What is an alternate to chromosome walking?
Physical map: Series of contigs that span the entire genome.
Alternatively to chromosome walking, you can use the minimum tile path and sequencing your entire region to reduce the issue of redundant clones. There are problems with gaps though where a computer cannot find clones to span a particular region. To overcome this you can digest you DNA with a restriction enzyme that cuts less often, thus generating larger fragments. You can then ligate these to create circular fragments, and then ligate the region at which the two ends joined, so you have regions that were far apart, no next to each other, and you can use this to overcome gaps by finding the appropriate fragments that hybridise to either side of this junction fragment. You still have a gap, but at least now you have your clones in the correct order and orientation.
What are some reasons to generate a physical map?
(i) Positional cloning/chromosome walking
(ii) Bridging gaps within genome assembly through paired end reads and sequence alignment
(iii) Source of potential markers and probes
(iv) Detailed analysis of a particular region, looking for TE’s, inversions, repeats or other structural variation to gain more insight into a region and its function.
What are Anchor Loci?
Anchor loci are highly conserved genes or markers that are single copy (so no ambiguity about genomic location) and unique in the genome (no redundancy). The fact that they are highly conserved means that you can use them as markers when comparing across different species, to look for things like gene rearrangements or chromosomal inversions. They allow you to see if synteny (same genes on same order) is conserved, along with colinearity (same genes in same order).
Given the requirements of anchor loci, they are often genes involved in major, highly conserved biological processes, such as DNA replication or translation or respiration.
Talk about how complexities in genomes make it harder to map
Differing degrees of complexities in genomes make it harder and harder to map genes to certain locations and to then compare these regions across multiple species, for whom the region may or may not be in the same genomic location, or same order or same copy number.
Genetic maps are achieved through linkage analysis and allow ordering of genes, until you reach genes that are too close such that they have a 0cM recombination frequency. Also, the problem with cM as a unit of measurement is that it is relative and hence can be potentially any distance small or large in physical units; also, cM’s are not additive.
You can however use genetic maps to isolate clones from genomic libraries and generate a series of overlapping clones that span a region of interest. There is the inherent problem of gaps here, but at least you have clones in the correct order.
Ideally you want a physical map that spans the entire region, without gaps.
Microsatellites
Genetic marker with short sequence length
2-9bp repeat unit
Detecting microsatellites
Flanking repeats are highly conserved
More repeats –> larger amplified fragment –> electrophoresed
Slippage model
In a highly repetitive region, difficult for replication machinery to determine what is already replicated –>
region may be ignored –> lose sequence OR
region replicated twice –> gain sequence
How to sequence conserved flanking regions
Form genomic library
Probe it for SSR
SSR BENEFITS
Score for more than one marker at a time(codominant)
Highly abundant & extreme levels of polymorphism
Can be used across all species
Small amount of DNA needed
INBREEDING
Mating between related individuals
Why does inbreeding lead to decrease in fitness of individuals?
- Increases likelihood of homozygosing deleterious recessive alleles
- Increases homozygosity such that it acts against heterozygote advantage
Inbreeding results in:
Decreased: fertility body size litter size overall health FEWER INDIVIDUALS PRODUCED
Inbreeding depression
Reduction in fitness due to inbreeding
Gambler’s rule/extinction vortex
decreased population numbers because of inbreeding –> more inbreeding –> even fewer individuals to mate with –> EXTINCTION
