Lecture 15

Question 1

Up the top of the gel:

Answer 1

• Large fragments

Question 2

Down the bottom of the gel:

Answer 2

• Smaller fragments

Question 3

What are we scoring?

Answer 3

• The presence or absence of bands

- We don’t know if this is hetero- or homo- zygous

Question 4

How do you map things with AFLPs?

Answer 4

• Maximum signal is obtained through maximum divergence

- Reduction of the noise, to ensure that A is homozygous and isogenic

Question 5

F1 will be hybrids between A and B. This can result in achiasmatic and chiasmatic individuals:

Answer 5

• Achiasmatic: No crossing over in meiosis in females (in butterflies)
• Chiasmatic: Crossing over in meiosis can happen in males (in butterflies)

Question 6

Identical segregation patterns indicate:

Answer 6

• 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

Question 7

How can you determine which are recombinants?

Answer 7

• Offspring with one band from the father (10kb) and one from the mother (either 100kb or 200kb)

Question 8

Combining genetic and physical data via common markers:

Answer 8

• 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?!

Question 9

Genomic library:

Answer 9

• A genetic library covering the whole genome with markers along it

Question 10

How do we close the gap between the twp=o markers?

Answer 10

• 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

Question 11

Contig:

Answer 11

• The overlapping clones covering a chromosomal region

Question 12

Physical map:

Answer 12

• A set of contigs that covers the entire genome

Question 13

Making a Bacterial Artificial Chromosome (BAC) library:

Answer 13

• 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

Question 14

Marker conversion applies to all kinds of DNA markers and uses this method:

Answer 14

• 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

Question 15

Overlapping clones must share something!

Answer 15

• In order for them to be informative

Question 16

Chromosome walking to see if you are walking in the correct direction

Answer 16

• 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)

Question 17

Chromosome walking, identifying the next clone:

Answer 17

• PCR amplify BAC end or an internal region, hybridise the amplicon to BAC library
• Identify overlapping BAC clones and repeat the process

Question 18

Minimum tile path:

Answer 18

• A library of clones is generated, but many of them are redundant as they overlap so much.
• The minimum tile path is the minimum number of clones needed in order to cover the entire genome
• Each tile can be shot gun sequenced to create 1kb sequences
• Can convert the BAC clone to in situ hybridising probes, so you can combine the physical map to the genetic map