CHAPTER 9: Digital Analysis of DNA

the haploid genome of Drosophila melanogaster contains approx. 120 million bp of DNA. if a genomic DNA library contains clones with an average size of 12 kb, how many clones would make up one genomic equivalent

1,000 clones (120,000,000/120,000)

place the steps involved in whole-genome shotgun sequencing in order: make a shotgun library of the entire genome isolate genomic DNA sequence the ends of each individual clone in the library identify overlaps between DNA sequences and use the overlaps to reconstruct the entire sequence

1. isolate genomic DNA 2. make a shotgun library of the entire genome 3. sequence the ends of each individual clone in the library 4. identify overlaps between DNA sequences and use the overlaps to reconstruct the entire sequence

restriction enzyme Notl recognizes a 8-bp DNA sequence and cuts genomic DNA every 65,500 bp, on average. If a haploid genome, which consists of about 3 billion bp, is digested with this enzyme, about how many restriction fragments will be produced

46,000 fragments

Notl is a restriction enzyme that recognizes the sequence 5`-GCGGCCGC-3`. make the simplifying assimption that a genome sequence has equal proportions of all four bases in random order. if this genomic DNA is digested with Notl, the average size of the resulting restriction fragments will be approx. _______

65.5 kb (1/4^8 = abt 65,500)

a group of identical DNA molecules are called a

DNA clone

lab technician using ultrasoud to shear DNA accidently used a level of ultrasound energy higher than she intended. what result would this mistake have on the size of the DNA fragments produced by ultrasound treatment?

DNA fragments would be smaller than expected

when cloning a gene into a vector, the sugar-phosphate backbone of each DNA molecule is covalently linked by what enzyme

DNA ligase

enzyme that catalyzes DNA replication

DNA polymerase

EcoRI site

DNA sequence that can be recognized and cut by a restriction enzyme

DNA library

a collection of cloned DNA molecules

recombinant DNA molecules

a combination of DNA molecules with parts having different origins that were joined using recombinant DNA technologies.

amp^R

a gene that makes a cell that contains the plasmid resistant to an antibiotic

shotgun strategy

a genome sequencing approach in which the clones that are sequenced are selected from a library at random

cellular clone

a group of cells that all descend from a single progenitor cell.

a small circular DNA molecule that can replicated within bacterial cells and is often used as a vector in gene cloning

a plasmid

electrophoresis

a process used to separate DNA fragments, RNA molecules, or polypeptides according to their size. Electrophoresis is accomplished by passing an electrical current through agarose or polyacrylamide gels. In response to the current, molecules migrate through the gel, and at different rates that depend on their sizes.

DNA clone

a purified sample containing a large number of identical DNA molecules.

two specialized DNA sequences a vector must have in order to be used as a cloning vector

a sequence that allows the vector to be detected by the investigator a sequence into which foreign DNA can be inserted

paired-end sequencing

a strategy for determining the base pair sequence of whole genomes in which ~1000 bp at both ends of single BAC clones are sequenced. Knowing that the two sequence reads are connected on a single BAC insert allows genome assembly despite the presence of repeated elements.

polylinker

a synthetic DNA sequence in a cloning vector containing several different restriction enzyme recognition sites that can be used for insertion of a DNA molecule.

a recombinant DNA molecule has covalently linked DNA fragments from...

a vector and an inserted fragment

if a restriction enzyme cuts a DNA molecule at the line of symmetry within its recognition sequence, it will produce DNA fragments with...

blunt ends

a colony is made up of genetically identical bacterial _______, and a DNA clone is made up of identical molecules of a(n) ________ within the colony

cells; plasmid

after bacterial cells have been transformed with foreign DNA and allowed to grow into a colony, the colony as a whole is called a(n) _________, while the many plasmid molecules within the colony make up a(n) ________

cellular clone; DNA close

molecular cloning is a two step process. the first, DNA fragments are inserted into a _____ _______, and then the resulting molecules are amplified inside of living cells

cloning vectors

a vector requires an origin of replication so that it can be...

copied many times by the host cell

in genetics, the term digestion refers to

cutting a DNA molecule with a restriction enzyme

a researcher would most likely choose to use a polyacrylamide gel rather than an agarose gel in order to...

distinguish DNA fragments with small size differences

the technique that is used to separate charged molecules based on their movement in an electric field is called

electrophoresis

after electrophoresis, DNA molecules can be visualized by incubating them with a fluroescent dye called _______ _________, washing away unbound dye, and then placing the gel under ultraviolet light

ethidium bromide

the number of clones that would be present in a genomic library containing exactly one copy of every locus in the genome is called a(n)

genomic equivalent

a collection of cellular clones containing copies of every sequence in an organism's genome is called a(n)

genomic library

if bacteria are transformed with a plasmid carrying an antibiotic-resistance gene, one would expect progeny of that cell to _______ when exposed to the antibiotic

grow

a vector is a DNA molecule that can be used to...

introduce a DNA fragment of interest into a host cell that can replicate it

main advantage of the whole-genome shotgun strategy over the hierarchical strategy is that the whole-genome shotgun strategy....

is much less labor-intensive because BAC clones need not be ordered prior to sequencing

place the steps involved in the heirarchical strategy for sequencing a genome.: determine the order of the BAC clones, and construct a minimal tiling path sequence each BAC clone isolate genomic DNA reconstruct the genomic DNA sequence construct a BAC library with large inserts

isolate genomic DNA construct a BAC library with large inserts determine the order of the BAC clones, and construct a minimal tiling path sequence each BAC clone reconstruct the genomic DNA sequence

role of the polylinker in a plasmid

it contains a number of different restriction sites

a researcher may use restriction enzymes to digest the DNA of an organism. the fragments of DNA are then ligated into many vectors. this collection of recombinant vectors is called a(n)...

library

after electrophoresis, the size of a DNA fragment can be estimated by comparing the distance the DNA fragment migrated to the distance traveled by a set of __________ fragment in another lane of the gel.

marker

a bacterial enzyme that adds methyl groups to specifc DNA sequences is called a(n)

modification enzyme

process of isolating a single fragment of DNA from a complex mixture and making many exact copies of it in a living cell is called

molecular cloning

a vector has only two specialized DNA sequences: one that gives it the ability to replicate in E. coli cell and one that allows scientists to insert foreign DNA into it. Can this vector be used as a cloning vector by a scientists working with human DNA?

no, because it will be impossible to detect cells that have acquired the vector

one type of genome shotgun sequencing uses paired-end sequencing which refers to...

obtaining two sequence reads from a BAC clone, one from each end of the insert

the DNA sequence in a vector that allows the replication enzymes of the cell to make lots of copies of the vector

origin of replication

site that allows the plasmid to be copied in a host cell

origin of replication (ORI)

when Celera scientists sequenced the human genome, they used randomly selected BAC clones and used each random BAC clone to obtain two sequences, one from each end of the insert. this approach is known as

paired-end sequencing

a DNA sequence in one strand that is identical when read in the opposite direction in the other strand is called a

palindromic sequence

DNA fragments that are subjected to electrophoresis will always move toward the positively charged electrode because the ______ ______ in the backbone of DNA are negatively charged

phosphate groups

the fraction of viral particles that enter and replicate the inside host bacterial cells is called

plating efficiency

molecular cloning

process by which a single DNA fragment is purified from a complex mixture of DNA molecules and then amplified into a large number of identical copies.

the function of a restriction enzyme that occurs naturally in bacterial cell is to...

protect the cell from viruses

shearing of DNA by mechanical stress, such as sonication or passing DNA through a needle at high pressure, cuts DNA _____-

randomly

enzymes that bind to a specific sequence in a double-stranded DNA molecule and cut the DNA backbone of each strand are called...

restriction enzymes

a DNA fragment generated by cutting a larger piece of DNA with a restriction enzyme is called a(n)

restriction fragment

to clones a DNA molecule, a researcher would digest the DNA of interest and a vector with the same _________ enzyme, then join the two molecules together with the enzyme ______ _______. finally, the recombinant molecule is introduced into bacteria through _______

restriction; DNA ligase; transformation

a resistance gene that allows a host cell containing a vector to grow on a toxic substance is called a(n)..

selectable markers

"sticky ends" created by cutting DNA with a restriction enzyme are...

single-stranded overhangs of DNA

the general analysis of base pair fragments through electrophoresis is that...

small base pair fragments travel further down the gel than large base pair fragments

place the steps involved in cloning a recombinant DNA molecules in the correct order: the recombinant DNA molecule is transformed into bacterial cell the DNA of interest and a vector are digested with the same restriction enzyme DNA ligase is used to join the DNA of interest and a vector bacterial cells are grown on medium containing a selectable marker, such as an antibiotic

the DNA of interest and a vector are digested with the same restriction enzyme DNA ligase is used to join the DNA of interest and a vector the recombinant DNA molecule is transformed into bacterial cell bacterial cells are grown on medium containing a selectable marker, such as an antibiotic

restriction

the bacterial capacity for limiting viral growth. the ability of bacterial cell to prevent the replication of a virus

what assumptions are made in order to estimate the average length of the fragments that will be produced by digestion of a piece of DNA with a restriction enzyme

the bases are randomly distributed in the DNA sequence and that the four bases occur in equal proportions

what is the purpose of including marker fragments in a lane of an electrophoresis gel?

the distance traveled by the marker fragments can be used to estimate the sizes of other fragments

genomic equivalent

the number of different DNA clones—with inserts of a particular size—that would be required to carry a single copy of every sequence in a particular genome.

modification

the phenomenon in which growth on a restricting host results in modification of a virus so that succeeding generation grow more efficiently on the same host strain

variables that affect the rate at which molecules move during electrophoresis

the strength of the electric field that is applied the composition of the gel the physical size of the molecule

processs by which a cell or organism takes up a foreign DNA molecule

transformation

what type of cell treatments are used to increase the likelihood that transformation will occur

treatment of bacterial cells with cold CaCl2 or subjecting them to eletric shock

selectable markers

vector genes that make it possible to identify cells harboring a recombinant DNA molecule.

YACs (yeast artificial chromosomes) & BACs (bacterial artificial chromosomes) are

vectors that can be used to clone large segments of DNA

cloning vectors

vehicles for introducing foreign DNA into host cells, where that DNA can be reproduced in large quantitites; a DNA molecule into which another DNA fragment of appropriate size can be integrated without loss of the vector's capacity for replication.

many species of bacterial cells make restriction enzymes to protect themselves from invasion by

viruses