Schneider Genetics Exam 3 - Chapter 10 and 11

1. Restriction enzymes cut the sugar-phosphate backbone of which DNA strand? 2. Roughly how many restriction enzymes now available?

1. BOTH strands 2. hundreds

Shotgun sequencing: 1. ________ developed the whole-genome shotgun strategy for genome sequencing 2. Their strategy was referred to as _________ sequencing

1. Celera 2. paired-end

1. _________ and ________ DNA sequence from automated Sanger sequencing 2. Computer reads of sequence complementary to the _______ strand 3. Sequence is read from ______ to _______

1. DNA sequence trace; inferred 2. template 3. left to right (5'-to-3' synthesis from primer)

Molecular cloning step 2: 1. _______ cells take up and amplify recombinant DNA 2. _______ is the process by which a cell or organism takes up foreign DNA

1. Host 2. Transformation

1. An accurate sequence of the human genome was completed in 2003 by the _________ 2. By 2021, the genomes 0.2% of all known animal species have been sequenced

1. Human Genome Project

1. Electrophoresis moves _________ in an electric field 2. DNA has _______ charge, so moves toward _______ charge

1. charged particles 2. negative; positive

RsaI is a restriction enzyme that recognizes a 4-bp sequence. It cuts genomic DNA, on average, every 256 bp. The haploid human genome consists of approximately 3 billion bp of DNA. If the haploid human genome is digested with RsaI, approximately how many restriction fragments will be produced? 256,000 fragments 700,000 fragments 11,800,000 fragments 46,000 fragments

11,800,000 Reason: 3,000,000,000/256 = 11,718,750 which is closest to 11,800,000.

Compared to a deoxyribonucleotide triphosphate (dNTP), a dideoxyribonucleotide triphosphate (ddNTP) is missing its ____. 3' hydroxyl group 2' hydroxyl group 1' nitrogenous base 5' phosphate group

3' hydroxyl group

The restriction enzyme BamHI recognizes the sequence 5'-GGATCC-3'. Making the simplifying assumptions that a genome sequence contains all four bases in equal proportions and in random order, what is the average size of the restriction fragments that will be produced by digestion of genomic DNA with BamHI? 1296 bp 256 bp 4096 bp 24 bp

4096 bp

Choose the two specialized DNA sequences a vector must have in order to be used as a cloning vector. Multiple select question. A sequence that allows the vector, and the foreign DNA inserted in it, to replicate A sequence that allows bacteria transformed with the vector to be detected by the investigator A sequence that specifies a restriction endonuclease A sequence that allows the synthesis of a protein from the inserted foreign DNA

A sequence that allows the vector, and the foreign DNA inserted in it, to replicate A sequence that allows bacteria transformed with the vector to be detected by the investigator

During electrophoresis, which of the following DNA fragments will travel the greatest distance from the well? A DNA fragment with a low GC content A small DNA fragment A large DNA fragment A DNA fragment with a high GC content

A small DNA fragment

True or false? Genomes of animals, plants, and microorganisms can be analyzed all at once

False Genomes are too large

What name is given to the entire complement of genetic material in an organism? Phenotype Genome Genotype Proteome Transcriptome

Genome

_________ are collections of cloned fragments

Libraries

Cloning with Plasmid Vectors: Step 2 ________ is used to seal the phosphodiester backbones between vector and insert.

Ligase

A vector has only two specialized DNA sequences: one that gives it the ability to replicate in E. coli cells 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 No, because the vector must be able to replicate in both E. coli and human cells Yes, because it will be relatively easy to isolate it from E. coli cells

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

Shotgun sequencing: What prevents correct assembly of single shotgun sequence reads?

Repeats

What process is catalyzed by the enzyme DNA polymerase? Replication of DNA Cutting of the DNA backbone at a specific sequence Ligation of two DNA molecules with identical sticky ends Transcription of DNA into RNA

Replication of DNA

Which of the following is an advantage of sticky DNA ends compared to blunt ends in molecular cloning? Sticky ends prevent DNA ligase from "closing" a plasmid without an insert. DNA ligase is more efficient at connecting two DNA strands. Single-strand overhangs are available for base pairing.

Single-strand overhangs are available for base pairing.

Why would one use a vector with a selectable marker? To create site-directed mutants To identify protein-binding DNA sequences To identify cells containing the vector To allow the enzymes of the host cell to replicate the vector

To identify cells containing the vector

What is the purpose of molecular cloning? To track the inheritance of an allele To determine what vectors are plasmids To analyze DNA binding proteins To produce many copies of a DNA molecule of interest

To produce many copies of a DNA molecule of interest

True or False? Each genomic DNA fragment can form a different recombinant molecule

True

True or False? In Sanger sequencing, once the dideoxyribonucleotide is created, no new ones can follow

True

True or False? Sticky ends produced with the same restriction enzyme are always compatible, regardless of DNA origin.

True

After electrophoresis, visualize DNA fragments by staining gel with fluorescent dye, and photograph gel under _________

UV light (lambda)

A complete genomic library contains _____. at least 95% of the sequences in an organism's genome a single copy of every sequence of an organism's genome a single copy of only the gene sequences in an organism's genome

a single copy of every sequence of an organism's genome

A recombinant DNA molecule has covalently linked DNA fragments from _____. genes that encode proteins and pseudogenes genes and intergenic genomic regions a vector and an inserted fragment

a vector and an inserted fragment

A genomic library is a collection of cellular clones containing copies of ______. only the coding sequences of genes from an organism each full-length chromosome from an organism all DNA sequences in the genome of an organism

all DNA sequences in the genome of an organism

Where on the genome does a restriction enzyme recognize a specific sequence of bases?

anywhere

The whole-genome shotgun approach can be highly _________.

automated

In ________ sequencing, fragments flow past a laser beam and the color of the terminal base is digitally recorded.

automatic

Transformation is the process in which _____. restriction enzymes cut the DNA backbone and create sticky ends cells take up foreign DNA molecules a virus is used as a vector instead of a plasmid a piece of chromosomal DNA is ligated into a plasmid vector

cells take up foreign DNA molecules

A collection of cellular clones that contain one (and only one) copy of every sequence in the entire genome of an organism is called a(n) _________ genomic library.

complete

A vector requires an origin of replication so that it can be ______. altered by the host cell copied many times by the host cell analyzed through DNA sequencing used to express proteins

copied many times by the host cell

Restriction enzymes are enzymes that can ____. cut the DNA backbone and reveal protein-binding sequences identify host cells containing vectors cut the DNA backbone at specific sequences create site-directed mutants

cut the DNA backbone at specific sequences

A nucleotide that lacks both the 2' and 3' hydroxyl groups is called a(n) _________.

ddNTP

Restriction fragments are generated by the ________ with restriction enzymes

digestion of DNA

To estimate the average restriction fragment size that will be produced by digestion with a restriction enzyme, it is convenient to assume that the four bases occur in _________ proportions and that they are distributed ________ in the DNA sequence.

equal; randomly

After electrophoresis, DNA molecules can be visualized by incubating them with a fluorescent dye called ____________ washing away unbound dye, and then placing the gel under ultraviolet light.

ethidium bromide (EtBr)

After electrophoresis, DNA may be incubated with a compound called ethidium bromide in order to _______. make the DNA fragments in the gel visible under ultraviolet light covalently attach the DNA molecules to the molecules that make up the gel matrix determine the sequence of the DNA fragments in the gel

make the DNA fragments in the gel visible under ultraviolet light

A researcher who wanted to break DNA at random locations would most likely subject the DNA to _______. methylation by a modification enzyme, followed by digestion with a restriction enzyme complete digestion with a restriction enzyme gel electrophoresis mechanical shearing forces

mechanical shearing forces

The 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

Restriction enzymes usually recognize ________ sequences in which the sequence in one strand is identical to the complementary strand read in the opposite direction

palindromic

Molecular Cloning Step 1: You can create recombinant DNA molecules with _________, which are circular DNA

plasmid vectors

The fraction of viral particles that enter and replicate inside host bacterial cells is called the ________ efficiency.

plating

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

The enzyme that catalyzes DNA replication is called __________.

DNA polymerase

Choose two important advantages of using sticky ends in molecular cloning. Multiple select question. Sticky ends ensure that DNA is always inserted into the plasmid (the plasmid cannot be circularized without an insert). Sticky ends can be connected without DNA ligase. Fragments with sticky ends have overhangs that can base pair. Sticky ends produced with the same restriction enzyme are always compatible, regardless of DNA origin.

Fragments with sticky ends have overhangs that can base pair. Sticky ends produced with the same restriction enzyme are always compatible, regardless of DNA origin.

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

selectable

The use of dideoxyribonucleotides with different colored fluorescent dyes allows the detection of the ______. appropriate vector amount of DNA sequence of DNA gene of interest

sequence of DNA

In automated Sanger sequencing, the 5' end are the _______ fragments and the 3' end are the _______ fragments

smaller; larger

Cloning fragments of DNA: 1. _______ cloning is a means to purify a specific DNA fragment away from all other fragments and make many identical copies of the fragment 2. There are two basic steps to the process: a.Insert DNA fragments into cloning vectors to make a _________. b.Transport this DNA into a _______ to be copied.

1. Molecular 2. a) recombinant DNA molecule. 2. b) living cell

Sanger sequencing: chain termination method 1. ________ fragments are separated by size using electrophoresis 2. A special gel can separate DNA fragments that differ in size by _______ nucleotide. 3. Smaller DNA fragments migrate quickly and appear at the _______ of the gel.

1. Nested 2. only one 3. bottom (or end or right side or whatever the ****)

Fragmenting DNA Method: Restriction Enzymes 1. _________ sites for restriction enzymes are usually 4 - 8 bp of double-strand DNA 2. These are often ________ which means the base sequences of each strand are identical when read 5'-to-3' (Like the name Hannah) 3. Each enzyme cuts at same place relative to its specific ________ sequence

1. Recognition 2. palindromic 3. recognition

Two methods of fragmenting DNA: 1. ________ that can precisely cut DNA, and ___________. 2. ________ causes random fragmentation and generates random sized fragments; ________ is fragmented at specific sites in DNA/sequence specific

1. Restriction enzymes; mechanical shearing 2. Mechanical shearing; restriction enzymes

Modern DNA sequencing methods: 1. _______ sequencing (chain termination method) is good for sequencing small pieces of DNA 2. ______ sequencing is good for whole genome sequencing

1. Sanger (sewing machine, precise small pieces, needles) 2. Shotgun (gun that shoots the whole genome)

Shotgun sequencing: Paired-end sequencing has three steps:

1. Shear DNA to make uncharacterized (shotgun) clones 2. Sequence from BOTH ends 3. Assembles based on overlapping

Sanger sequencing: chain termination method 1. Incorporation of a _________ terminates DNA synthesis 2. That's because ddNTP lacks a _______

1. dideoxynucleotide (ddNTP) 2. 3' -OH

Sanger sequencing: chain termination method 1. Each ddNTP is labeled with a different ________ for detection of the sequence 2. Each lane displays the sequence obtained from a separate DNA sample and _______. 3. Each fragment has ________ is labeled with a specific fluorescent dye.

1. fluorescent dye 2. primer 3. terminated with a specific ddNTP

1. A _________ is a long-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector 2. Each colony contains a different __________, each with a part of the human genome.

1. genomic library 2. recombinant plasmid

1. The Human Genome Project began using the ________ strategy of sequencing 2. was it successful?

1. hierarchical 2. yes but expensive and labor intensive

steps involved in whole-genome shotgun sequencing: 1. ________ genomic DNA. 2. Make a ________ of 500-1000bp fragments from the entire genome in plasmid vectors. 3. Sequence the ________ of randomly chosen library plasmids. 4. Use a ________ to identify overlaps between DNA sequences and assemble progressively larger contigs.

1. isolate 2. genomic library 3. inserts 4. computer

1. With _______ DNA fragments, migration distance through gel depends on size. 2. Determine size of unknown fragments by comparison with _________ of known size.

1. linear 2. migration of DNA markers

Sanger sequencing: chain termination method After fragments are separated, two things are determined:

1. number of bases in the chain 2. last base in the chain

A cloning vector must have two specialized sequences:

1. one that can replicate in the target cells and allows allows scientists to insert foreign DNA into it. 2. one to signal the vector's presence

Features of the plasmid vector: 1. Site that allows the plasmid to be copied in a host cell is the _________. 2. A gene that makes a cell that contains the plasmid resistant to an antibiotic is _______. 3. DNA sequence that can be recognized and cut by a restriction enzyme is the _______.

1. origin of replication 2. ampR 3. EcoRI site

Fragmentation of DNA method: Mechanical Shearing 1. Mechanical forces can break ________. 2. Process can be passing DNA through a thin _______ at ______ pressure or the process of _______ 3. ______ can be blunt, or may have protruding single-stranded regions.

1. phosphodiester bonds 2. needle; high; Sonication (ultrasound energy) 3. Ends

Plasmid cloning vectors have three main features: 1. Origin of _______. 2. Restriction site(s) for cloning _______ DNA 3. A ________ (e.g. antibiotic resistance) (Gene that produces a protein that allows resistance

1. replication 2. insert 3. selectable marker

Sanger sequencing: chain termination method 1. Sanger generates a series of _______-stranded DNA fragments 2. DNA fragments include the primer and nucleotides complementary to the ______ DNA. 3. The DNA fragments are a _________, which means they each differ in length from the proceeding and succeeding fragment by one nucleotide

1. single 2. unknown 3. nested array

Sanger sequencing: chain termination method 1. You want the fragments to range from 4-500 stopping at every step (all 500) to get different _______ and _______. 2. You read the color code from _____ to _____ 3. What would happen if you had no termination?

1. sizes and different last added letters (because of color) 2. bottom to top 3. polymerase would synthesis the entire fragment

(Shotgun sequencing) Paired-end sequencing: 1. Pairs of sequence give ________ information 2. Paired-end sequences direct correct assembly of unique sequences flanking ________

1. spatial **e.g. two ends of a 200 kb insert are 200 kb apart in the genome 2. repeats *a way to get around the repeat problem

Sanger sequencing: chain termination method Components required: 1. The _______ is the single strand of DNA to copy 2. __________, or the building blocks of DNA 3. The ________ is the short single stranded DNA molecule that is complementary to the template short sequence that's just like replication 4. The Special Ingredient: ___________ (ddATP, ddCTP, ddGTP, ddTTP)

1. template 2. Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP) 3. primer 4. Dideoxyribonucleotide triphosphates

The restriction enzyme NotI recognizes a 8-bp DNA sequence and cuts genomic DNA every 65,500 bp, on average. If a haploid human genome, which consists of approximately 3 billion bp, is digested with this enzyme, approximately how many restriction fragments will be produced? 12,000,000 fragments 46,000 fragments 65,500 fragments 700,000 fragments

46,000 fragments

NotI is a restriction enzyme that recognizes the sequence 5'-GCGGCCGC-3'. Make the simplifying assumption that a genome sequence has equal proportions of all four bases in random order. If this genomic DNA is digested with NotI, the average size of the resulting restriction fragments will be approximately ______. 65.5 kb 250 bp 1.0 kb 4.1 kb

65.5 kb

NotI is a restriction enzyme that recognizes the sequence 5'-GCGGCCGC-3'. Make the simplifying assumption that a genome sequence has equal proportions of all four bases in random order. If this genomic DNA is digested with NotI, the average size of the resulting restriction fragments will be approximately ______. 65.5 kb 250 bp 4.1 kb 1.0 kb

65.5 kb 4^n

What is a restriction fragment? A protein domain within a restriction enzyme A gene that encodes a restriction enzyme A DNA sequence that can be recognized by a restriction enzyme A fragment of DNA generated by a restriction enzyme

A fragment of DNA generated by a restriction enzyme

What is a plasmid? A virus that is missing several genes and is often used as a vector in gene cloning A large linear DNA molecule with an origin of replication A gene for a selectable marker, such as antibiotic resistance A small circular DNA molecule often used as a vector in gene cloning

A small circular DNA molecule often used as a vector in gene cloning

_________ artificial chromosomes and ________ artificial chromosomes are alternate cloning vectors that can carry ______ inserts.

Bacterial; yeast; large

True or False? Since humans are so complex, we have the largest genome size

False We are outgenomed by the lungfish and the canopy plant (largest known)

Which of the following 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? Multiple select question. The bases are randomly distributed in the DNA sequence. The four bases occur in equal proportions. All genes are the same length. Restriction enzyme recognition sequences are always the same distance away from one another in the genome.

The bases are randomly distributed in the DNA sequence. The four bases occur in equal proportions.

In genetics, the term digestion refers to ______. cutting a DNA molecule with a restriction enzyme identifying the genes within a large segment of DNA creating a specific mutation in a molecule of DNA introducing a piece of foreign DNA into a host cell

cutting a DNA molecule with a restriction enzyme

Plating efficiency is the ______. fraction of viral particles that enter and replicate inside host bacterial cells rate at which a laboratory technician can prepare petri dishes rate at which a DNA fragment travels through an agarose gel during electrophoresis fraction of bacterial cells that survive being transferred from one petri dish to another

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

A collection of DNA fragments of various lengths was subjected to electrophoresis. Arrange these DNA molecules in order, based on the distance they would travel from the well. The molecule that travels the shortest distance should be at the top of the list, and the molecule that travels the greatest distance should be at the bottom of the list: 6000 bp, 8500 bp, 4200 bp, 900 bp, 2500 bp

from shortest to longest distance: 1. 8500 bp 2. 6000 bp 3. 4200 bp 4. 2500 bp 5. 900 bp

The process of _________ distinguishes DNA fragments according to size

gel electrophoresis

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

gel electrophoresis

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

genomic library

Restriction enzymes usually recognize what kind of sequences?

palindromic

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

phosphate

The products of a Sanger sequencing reaction can be separated from one another through the use of __________ gel electrophoresis.

polyacrylamide

During electrophoresis, DNA fragments move toward the electrode with a(n) ________ charge because the phosphate groups in the backbone of DNA each carry a(n) _________charge.

positive; negative

Shearing of DNA by mechanical stress, such as sonication or passing DNA through a needle at high pressure, cuts DNA ______. at the ends of chromosomes at specific DNA sequences between genes randomly

randomly

A molecule that has covalently linked DNA fragments from at least two sources is called a _____________ molecule.

recombinant DNA

The primer component of Sanger sequencing is just like __________.

replication

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

restriction fragment

To clone 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 DNA _________. Finally, the recombinant molecule is introduced into bacteria using a process called __________.

restriction; ligase; transformation

"Sticky ends" created by cutting DNA with a restriction enzyme are ______. single-stranded overhangs of DNA sequences that confer antibiotic resistance sequences that can be used to initiate replication of a DNA molecule

single-stranded overhangs of DNA

When a collection of DNA fragments is separated by electrophoresis, different DNA fragments will migrate at different speeds due to differences in their _________.

size **length, mass, or molecular weight

Cloning with Plasmid Vectors: Step 1 Digestion of the vector and human genomic DNA with a restriction enzyme results in complementary ________.

sticky ends

The general ideas behind genome sequencing are simple:

•Fragmenting the genome •Cloning DNA fragments •Sequencing DNA fragments •Reconstructing the genome sequence from fragments breakin it apart, copying DNA, sequencing DNA, rebuilding it from parts