genetics quiz 6

human genome project

international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping, and sequencing all the genes of the human genome from both a physical and a functional standpoint

What did the human genome project start and when was it completed?

started in 1990 and was completed in 2003

by 2019, the genomes of approx ___ (eukaryotic) and approx ___ (prokaryotic) species have been sequenced

8000, 200000

reference genome

a full sequence that best represents the genome of a speciees

What can we learn from sequencing and comparing genomes?

• functions of the human genome

• how the genomes differ in number of genes

• what a genome sequence tells us about evolutionary relationships between species

• how genomes evolve over time

Are the genomes of elephant sharks or tiger tail sea horses slower evolving?

elephant sharks have a slower evolving genome than tiger tail sea horses

What were the keys to success of the human genome project?

• embrace partnerships (worldwide collaborations, multiple disciplines)

• spur technology (tech evolved)

• harness the data (computers and genetics)

• share data

• address societal concerns

• applications

4 general ideas behind genome sequencing

• fragmenting the genome

• cloning DNA fragments (isolate and amplify) = recombinant DNA molecules

• sequencing DNA fragments

• reconstructing the genome sequence from fragments (computer programs)

How can DNA be fragmented? (2 ways)

• digesting with restriction enzymes

• mechanical shearing of DNA

What do restriction enzymes do?

recognize and cut a specific sequence of bases (generally 4-8 bp of double strand and cut both strands) anywhere within the genome

digestion

when restriction enzymes cut 2 sugar phosphate backbones (phosphodiester), one of each strand

restriction fragments

fragments of DNA generated by restriction enzyme digestion

characteristics of restriction sites

• generally 4-8 bp of double strand

• often palindromic - base sequences of each strand are identical when read 5’ to 3’

blunt ends (restriction enzymes)

cuts are straight through both DNA strands at the line of symmetry

sticky ends (restriction enzymes)

cuts are displaced equally on either side of line of symmetry

Who won the Nobel Prize for the discovery of restriction enzymes and what observation led them to this belief?

• Werner Arber, Daniel Nathans, and Hamilton O. Smith

• viruses could grow abundantly in one bacterial strain but poorly in another strain of the same species

• this wasn’t hereditary

assumptions for estimating length of cuts

• genome is composed of 25% of each base (A, T, C, G)

• bases are randomly distributed

formula for estimating the length of cuts

4^n where n is the number of bases in the site

How can you produce DNA fragments of different lengths?

use different restriction enzymes that recognize sequences of different lengths

How do you calculate how many fragments of DNA will be produced from a restriction enzyme?

number of bp in haploid genome/average length of cut from RE

___ can be used to fragment DNA at random locations

mechanical forces

2 examples of mechanical forces that can break phosphodiester bonds

• passing DNA through a thin needle at high pressure

• sonication (ultrasound energy)

electrophooresis

movement of charged molecules (DNA fragments) in an electric field

steps of gel electrophoresis

• pour heated molten agarose into an acrylic plate to which a comb has been attached, allow to cool and harden

• remove comb, place gel in buffered aqueous solution, load DNA samples into wells in gel

• apply electric current (DNA has a negative charge, so moves toward positive charge)

• remove gel from tank after electrophoresis

• visualize DNA fragments by staining gel with fluorescent dye, photograph gel under UV light

with linear DNA fragments, migration distance through gel depends on ___

size

How can you determine size of unknown fragments in gel electrophoresis?

by comparison of migration to DNA markers of known size

molecular cloning

to purify a specific DNA fragment away from all other fragments and make many identical copies of the fragments

2 basic steps of cloning DNA

• insert DNA fragments into cloning vectors; ensure transport, replication, and purification of DNA inserts

• transport recombinant DNA into living cells to be copied

DNA clone

group of replicated DNA molecules (all identical)

Where are DNA clones stored?

in libraries within cells

3 main features of plasmid cloning vectoors

• origin of replication

• a selectable marker gene (for example antibiotic resistance)

• a synthetic polylinker, DNA sequence containing multiple restriction enzyme sites

alternate cloning vectors

• bacteria artificial chromosomes (BAC, 300kb) and yeast artificial chromosomes (YAC, 2000kb)

• can carry larger inserts than plasmids (20 kb)

digestion of the vector and human genomic DNA with a restriction enzyme results in ___

complementary sticky ends

___ is used to seal the phosphodiester backbones between vector and inserted fragment

DNA ligase

transformation

the process by which a cell or organism takes up foreign DNA

What percentage of E. coli cells will be transformed with plasmid?

0.1%

Which cells will grow on media with ampicillin?

only cells with plasmid

each cell with plasmid will produce a ___ on agar plate, the millions of identical plasmids in colony are a ___

colony, DNA clone

DNA purification and sequencing steps

• pick colonies

• inoculate

• incubate bacterial culture

• centrifugation

• resuspend cells

• lyse cells

• neutralize lysate

• separate cell debris

• bind plasmid DNA to matrix

• elute plasmid DNA

genomic library

long lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector

hierarchical approach to cloning

• break the genome into large fragments (BACs)

• map each BAC to a known chromosomal position

• sequence each BAC individually

• assemble BACs in order to reconstruct chromosomes

pros and coons of hierarchical approach

• pros: very accurate

• cons: very slow and expensive

Who began using the whole genome shotgun approach? What was the company name?

J. Craig Venter, Celera

general way that the whole genome shotgun approach works

uses cloning and sequencing of fragments of randomly cut DNA followed by assembly into a single continuous sequence

steps of whole genome shotgun approach

• create genomic library for overlapping fragmentation in plasmid vectors

• sequence DNA inserts of randomly chosen library plasmids all at once

• assemble sequences based on overlap of sequences into contigs

contigs

continuous base pair sequences

What technology is needed for the whole genome shotgun approach?

powerful algorithms and massive computing

What makes whole genome shotgun approach more effective than hierarchal approach?

• no need to BAC map first

• no clone by clone sequencing

Why had assembling the genome for a human not been done before Celera?

• human genome is full of repetitive DNA

• it was thought that without a physical map, repeats will make correct assembly impossible

Celery showed that human genome sequencing was possible thanks to…(3 things)

• very deep coverage

• paired end reads of different sizes

• advanced assembly algorithms

What’s different about the next generation sequencing techniques, compared to other approaches?

• don’t require a cloning step (adapters at the end of the fragments added instead, with index sequence to identify the sample)

• facilitated a metagenomics approach, in which DNA from a group of species in an environmental sample is sequenced

4 basic steps of next generation sequencing

• sample presentation

• cluster generation

• se

• data analysis