Flashcards in Lecture 15 Deck (18):
Up the top of the gel:
- Large fragments
Down the bottom of the gel:
- Smaller fragments
What are we scoring?
- The presence or absence of bands
- We don't know if this is hetero- or homo- zygous
How do you map things with AFLPs?
- Maximum signal is obtained through maximum divergence
- Reduction of the noise, to ensure that A is homozygous and isogenic
F1 will be hybrids between A and B. This can result in achiasmatic and chiasmatic individuals:
- Achiasmatic: No crossing over in meiosis in females (in butterflies)
- Chiasmatic: Crossing over in meiosis can happen in males (in butterflies)
Identical segregation patterns indicate:
- A linkage group!
- A linkage disequilibrium barcode can define the gene controlling the phenotype to the linkage group
- We still don't know how far away they are from each other
How can you determine which are recombinants?
- Offspring with one band from the father (10kb) and one from the mother (either 100kb or 200kb)
Combining genetic and physical data via common markers:
- Identify an interval surrounding your gene, and flank with two markers. You don't know how big the interval is or how many genes are within this region
- We must connect the genetic map to the physical map. How?!
- A genetic library covering the whole genome with markers along it
How do we close the gap between the twp=o markers?
- Identify overlapping genomic clones, by attempting to walk toward the other marker
- Find the overlapping sequences is a challenge
- Walking in the correct direction is another challenge
- The overlapping clones covering a chromosomal region
- A set of contigs that covers the entire genome
Making a Bacterial Artificial Chromosome (BAC) library:
- Isolate high molecular weight DNA
- Random shearing or partial restriction digest
- Select 100-150 kb fragments
- Clone into a vector
- Pick random colonies
- Develop a colony array
- And screen this array
Marker conversion applies to all kinds of DNA markers and uses this method:
- Cut an AFLP band from the gel
- Re-amplify using PCR
- Sequence the product and sequence the tagged site (STS)
- The PCR product can be used as a DNA probe to screen gDNA library, or the DNA sequence and its genomic location can be compared to other species
Overlapping clones must share something!
- In order for them to be informative
Chromosome walking to see if you are walking in the correct direction
- Develop new markers based on BAC sequence and see how far they are from the target locus, to make sure you are walking in the correct direction
- Sequence the ends and design new primers to amplify the parents of the mapping cross (there must be a polymorphism in order to design assays to genotype progeny to allow linkage analysis)
Chromosome walking, identifying the next clone:
- PCR amplify BAC end or an internal region, hybridise the amplicon to BAC library
- Identify overlapping BAC clones and repeat the process