Ch 20 Genomics I: analysis of DNA

Question 1

Genome

Answer 1

total genetic composition of organism or species

Question 2

Genomics

Answer 2

molecular analysis of an entire genome of a species

Question 3

Functional Genomics

Answer 3

examining genes to discover how thye interact to produce traits of an organism

Question 4

Structural Genomics

Answer 4

looking at proteins to see how genes interact to produce traits of an organism

Question 5

Proteomics

Answer 5

analysis of proteins of an entire genome

Question 6

Mapping

Answer 6

figuring out relative location of genes or segments of DNA on a chromosome

Question 7

Three ways to determine the organization of a genome?

Answer 7

Cytogenic Mapping, PHysical mapping, linkage mapping

Question 8

Cytogenetic Mapping

Answer 8

Determines location of gene along intact chromsome

Question 9

In what instance is cytogenetic mapping used?

Answer 9

With eukyotes because they have large chromsomes that can be looked at microscopically and individually categorized based on banding pattern of chromsomeos

Question 10

How accurate is cytogenetic mapping

Answer 10

Not super accurate becase it is only as good as the resolution of the microscope. Limit of approx. 5 mil base pairs. This is like a preliminary step for locating genes before physical mapping is used.

Question 11

FISH

Answer 11

Flourescence in situ hybridization

-Used to detect location of gene on chromsomes. that is HELD IN PLACE.

Question 12

How does FISH work?

Answer 12

1) Treat chromsomes of interest with agent that makes them swell and FIX to slide
2) Denature chromsomes
3) Add SINGLE STRANDED dna probes that have biotin
4) Add floresently labeled avidin that bind to biotin
5) View with microscope
* USually have two spots at each location because bound to both chromatids!

Question 13

Molecular Markers

Answer 13

Small segements of DNA that have unique qualities that can be seen under microscope or recognized by PCR and gel electrophoreses. Serve as guidlines along chromsomes to locate relative position of other genes

Question 14

Polymorphic Molecular Markers

Answer 14

Used in analysis of pedigrees to find mutant genes causing diseases

Question 15

How are restriciton enzymes used in physical mapping?

Answer 15

Used to create restriction fragments. Recognize palindromes and break chromsome into many smal pieces depending on spacing of palindromes

Question 16

How does random mutation effect palindromes?

Answer 16

In can either create or destroy palindromes

Question 17

EcoRi

Answer 17

Example of a restriction enzyme that recognizes 5-GAATC and digestes the chromosome into small pieces respectivly

Question 18

RFLP

Answer 18

Restriction fragment length polymorphism. Looking at chromsomes of different individuals in a pop. and seeing different lengths of restriction fragments due to loss or gain of palindromes

Question 19

Sourthern Blotting uses

Answer 19

used to deterime polymorphism fragments

Question 20

Microsatellites

Answer 20

Also reffered to as STR’s (Short tandem repeats) are short repetitive sequences interspersed throughout the genome

Question 21

Most common microsatellite in humans?

Answer 21

CAn (n is anywhere from 5-50 bp)

*found in every 10,000 base pairs

Question 22

What is sequece Tagged Site and how is it made?

Answer 22

Amplified microsatellite

*amplified using PCR

Question 23

How are STS sites made in detail?

Answer 23

STS is made using PCR.

1) add PCR primers that specifically target a sequence of a particular chromsome.
2) Many cycles of PCR produce the DNA fragment between the two primers.
3) Use gel electrophoresis that shows the microsatellites

Question 24

What are the two types of STS instances?

Answer 24

In homozygous individuals two microsatellites will be produced that are identical in length.
In Heterozygous the microsatellites might differ in length

Question 25

Physical mapping

Answer 25

requires cloning of several peices of DNA and presence of contigious series that overlap in genes

Question 26

How are chromosomal fragment categorized in physical mapping?

Answer 26

Based on length (bp) and the genes that are contained in said fragment

Question 27

Contig

Answer 27

a collection of clones containing overlapping pieces of DNA

Question 28

YAC, BAC, PAC

Answer 28

used to clone large segments of DNA used for eukaryotes

Question 29

How large of chrosomes can YAC take?

Answer 29

several hundred thousand to 2 million base pairs.

Question 30

What were BACS and PACS developed from and how many bases can they handle?

Answer 30

developed from F factors and P1 bacteriophage

Can handle 300,000 base pairs

Question 31

What is a cosmid?

Answer 31

A hybrid between a plasmid vector and a phage lambda. They except smaller pieces of DNA

Question 32

Positional Cloning

Answer 32

Cloning of gene based on its position along the chromosome

Question 33

Chromosome Walking

Answer 33

Used for positional cloning.

Question 34

High throughput sequencing

Answer 34

Sequencing DNA very rapidly

Question 35

Next Generation Sequencing Technology

Answer 35

New high throughput sequencing methods

Question 36

Second Generation

Answer 36

uses PCR to amplify large regions of DNA

Question 37

Third Generation

Answer 37

read single DNA molecules

Question 38

Pyrosequencing

Answer 38

An example of next generation sequencing

Question 39

How does Pyrosequencing work?

Answer 39

1) isolate DNA and break into fragment
2) Covalently attach oligonucleiotide adaptors to the 5 and 3’ ends
3) denature DNA into SINGLE STRANDS and attach to bead via the ADAPTORS
4) Emulsify beads so only ONE BEAD PER DROPLET
5) Beads put in picotiter plate. ONE BEAD PER WELL
6) Add sequencing reagents (DNA polymerase, primers, ATP sulfurylase, luciferase, apyrase, adenosine 5 posphosulfate and luciferin)
7) Flow A or T or G or C into each well.
8) When that base adds, a d light will appear

Question 40

Sequencing by Synthesis

Answer 40

involves identification of each nucleotide immediatly after incorporation ointo a DNA strand by DNA poly

Question 41

Comparitive Genomics

Answer 41

uses information from genome project to understand the genetic variation amond diff. pops.