Exam 3 Part 4 Flashcards Preview

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Flashcards in Exam 3 Part 4 Deck (20)
1

Why sequence genomes?

understand genetic variation with respect to phenotypic variation
inheritance
comparison to other organism's genomes
forensics
undertand genetics of extinct species
give insight into norml functions of genes
pharmacogenomics

2

Whole genome shotgun approach

1. Extract DNA from cells
2. cut DNA into small, overlapping fragments using restriction enzymes (performed in suboptimal conditions so not all sites cut)
3. clone contigs to make a genomic library
4. sequenced each clone using Sanger sequencing
5. used computers to reassemble sequences of contigs by puzzling together overlapping sequences
6. Deposited sequence information into NCBI bank

3

Sanger method

aka dideoxy sequencing, chain terminating
based on PCR of DNA template

4

If you don't know sequence, how can you design a primer?

1. can't design a primer against an unknown sequence
2. can use a universal primer and the same one

5

contigs

DNA fragments of various sizes (overlapping)

6

ddNTP

dideoxynucleic acid
Only has H instead of OH on 3' so phosphodiester bond cannot continue adding and synthesis stops

7

After reaction is complete

an array of products with flourescent molecules attached are separated by size using capillary gel electrophoresis

8

reassembling sequence

repeat sequencing for each clone in library
reassemble contigs using overlapping sequences

9

What we have learned

sequences of other organisms
3.2 billion baspairs
about 20,000 protein coding genes
5,000 genes do not code for protein
introns are large
genome is only 2% genes
average gene is 3,000 bp
genes are clustered together on chromosomes
99.9% sequence in common with other people

10

largest gene

dytrophin -2.4 million bp

11

What we haven't learned

long stretches of repeated DNA sequence that wre hard to reassemble
genes vs pseudogenes vs dubious ORFs
what some gene products actually do

12

How we find protein coding genes vs all other sequences

compare cDNA library to genomic library
use computer algorithms to look for consensus sequences

13

annotation

identification and description of genes and their important sequences
goal to assign functions to all of the genes in an organism
used to understand variation within an among organisms
identify where traits come from

14

Next generation sequencing

extract DNA
cut into contigs
affix DNA to solid support
one by one, wash DNA with dNTPS
If the known dNTP is incorporated, light emmits
reassemble using overlapping sequences

15

DNA marker

a specific region of DNA that varies among individuals
used to create a detailed map of the individual's genome

16

haplotype

set of SNPs that are close together on a chromosome
they are rarely scrambled by genetic recombination in a family

17

haplogroup

group of individuals that share a common ancestor bc they all have similar haplotypes

18

tag SNP

SNP used to represent an entire haplotype

19

SNP chips

more than 13 million human SNPs
allow for analysis of 1000s SNPs at once

20

Haplotype map

hapmap
a collection of all the combinations of haplotypes present in a population
used to study inheritance of complex traits
used to study evolutionary relatedness