Lecture 10 Flashcards
Hierarchal approach
start with 3 mil base pairs, chunk it to 200,000 and clone those chunks with bacterial artificial chromosomes, sequence and look for overlapping sequences, shotgun it at 500 bp
-slow method that uses BAC intermediates
-repetitive sequences wont affect this
shotgun
6 mil base pairs and shotgun the whole thing
-this approach works well for small bacteria bc smaller have less repetitive sequences
-it is bad if there is a repetitive sequence
-sanger
anotating sequence
editing and analyzing data
what is half the human genome?
repeating sequences
What percent of RNA is transcribed to RNA
28%
-only 1.1 to 1.4 is transcribed to proteins
ORF
protein encoding genes
-25,000
-starts at the start codon
-DNA/ RNA sequence without stop codon
theory supporting evolution?
most genes are in all of life and there are only a few differences in things like vertebrates
-2 people are 99.9% genetically identical
-1 in 1250 nucleotides are different
mRNA will be identical to what strand?
the sense strand
where are the genes that encode?
on both strands
-we can also have genes on the opposite strand that encode backward in the 5 to 3 direction
-each gene can encode multiple proteins
how can small genomes encode?
they can encode the same DNA chunk in the opposite direction at the same time
Proof the recombination frequencies are not uniforma cross a chromosome?
the closer the least likely theyare to separate and recombine
do more genes make an organism more complex
no they dont
-worms have more genes than people
-complexity is due to gene regulation (how the genome is organized)
how do we know where genes are
after examining many mutations we can build recombination frequency based on genetic map of where genes are in respect to each other. this is a classical sturtecantian genetic map but we cant do this with people because we would need families with thousands of kids
Do all ppl w BRACA 1 mutation have the same mutaiton?
no, they all have a different mutation at BRACA 1
methods of human gene mapping
-genome wide association screen
-positional cloning: can happen in a family
positional cloning
frequency between DNA markers and causative allele
-goal is the find gene marker so you can measure the distance between the mutation and the gene which will let you isolate the mutation, sequence it or analyze it for a pattern of expression
polymorphism
a sequence that varies between individuals in a population
-some of the sequences will have phenotypes some will be random and do nothing
-the variations are DNA markers
-can be SNP or SSR
genome wide association screening
a large population not closely related
-lets us look at the association between DNA marker and causative allele
-basically what marker the affeted individuals share
SNP polymorphism
-single nucleotide polymorphism
-ex: at the same site on person has a T and one person has a G while everything else is the same
SSR polymorphism
-simple sequence repeat (microsatellite)
-a repeating sequence that repeats a different amount of times in different people
does the marker cause the problem?
NO it just helps you find it
what happens to the marker as generations pass?
mutations will always be near the same polymorphism and may become linked to the polymorphism
How can we detect SSR?
PCR
-the SSR can be long (many repeats) or short
-PCR primers will be on both ends of the SSR
-you can be homo for long or short SSR marker or hetero
-we can also use a southern blot
does the marker cause the phenotype?
NO
How can we detect SNP
RFLP
-use a DNA probe that complements the SNP region
Microarray
chip w spots that show DNA position/ gene location
-for polymorphism= infinium
-we aneel the sequence to the array and add flourecent ddNTP
-can give genotype of the whole genome
RFLP
-cut the DNA w enzymes where the SNP is
-compare the lengths of the fragments w gel and look for location of SNP
Can a polymorphism be outside the coding sequence?
yes it can also be in the UTR or intron
the smaller the population
the less reliable the data is
-accuracy of data is based on LOD score
-increase LOD increase accuracy
