chapter 10 terms

___ amplifies small amounts of DNA into larger quantities for further analysis

PCR

in electrophoresis, the sample is added into the wells and the DNA fragments move toward the _____ end and are separated by what?

postive size (larger move slower and tend to be closer to the wells, the smaller dna fragments move faster towards the positive end)

Cas9 enzyme is found in what organsism

streptococcus pyogenes

why is the DNA gel electrophoresis transferred to the nylon filter?

to transfer the DNA in order to add the radioactive probe and then examined under xray

There are three recent methods of genetic editing. which of the following can target any of the 64 nucleotide *triplets* and can chain them together zinc finger nucleases TALEN CRISPR/Cas

zinc finger nucleases

what are the essential components of the PCR rxn

- DNA primer (initiates amplification) - Taq polymerase (heat stable) - double stranded DNA - gene of interest - deoxyribonucleotides

what are in the ingredients required in sanger sequencing

- DNA template strand - DNA primer - DNA polymerase - 4 standard deoxyNTP (dATP, dGTP, dCTP, dTTP) - *labeled* didoxyNTP (terminates strand)

what is the purpose of the southern blot

1) to look for the specific nucleotide sequence in the DNA being tested 2) identify the size of the fragments

useful vector has: (know)

1. ORI to initirate replication by dna polymerase 2. ability to carry suitable payload of dna 3. genes fro drug resistance to facilitate selection of cells containing plasmids

three basic steps of PCR technique

1. denaturation - heat, strand separate 2. priming- cools down then primer attach to ends of template strand, or *amplicon* 3. extension - DNA polymerase (Taq) synthesize complementary strand repeat

molecular cloning usually requires what three things

1. donor - desired donor gene is selected, excised by restriction endonuclease, and isolated 2. vector - gene inserted into a vector to transfer the gene 3. host - vector inserts gene into host

1. ______ cuts DNA precisely 2. ______ amplifies and increases number of copies 3. _______ produces visible records of specific nucleotides 4._____ locates specific loci & alleles

1. restrictive endonuclease 2. PCR 3. southern blot technique 4. hybridization

Place the following steps used in Southern blotting in the correct sequence: -1. Nitrocellulose sheet is placed against gel for transfer of DNA. -2. Nitrocellulose hybridization pattern is detected from marker (X-ray, fluorescence, etc.) -3. The nitrocellulose membrane is exposed to a labeled hybridization probe (DNA fragment). -4. Fragments from digestion of DNA are separated by gel electrophoresis. -5. Restriction enzymes are used to cleave (fragment) DNA.

5, 4, 1, 3, 2 -5. Restriction enzymes are used to cleave (fragment) DNA. -4. Fragments from digestion of DNA are separated by gel electrophoresis. -1. Nitrocellulose sheet is placed against gel for transfer of DNA. -3. The nitrocellulose membrane is exposed to a labeled hybridization probe (DNA fragment). -2. Nitrocellulose hybridization pattern is detected from marker (X-ray, fluorescence, etc.)

The steps involved in the Southern Blot test should be performed in the following order 1 = x-ray film 2 = electrophoresis 3 = digestion with restriction enzyme 4 = ethidium bromide 5 = radioactive probe A) 3, 2, 4, 5, 1 B) 3, 4, 2, 5, 1 C) 3, 2, 5, 4, 1 D) 2, 4, 3, 5, 1

A) 3, 2, 4, 5, 1

In the process of cloning eukaryotic DNA into prokaryotic cells, the role of reverse transcriptase is to make A) double-stranded eukaryotic cDNA from mature mRNA B) mature mRNA from precursor mRNA C) bacterial DNA from eukaryotic DNA D) double-stranded DNA with introns added back E) mRNA from DNA

A) double-stranded eukaryotic cDNA from mature mRNA

Many genetic engineering technologies: A. utilize gene probes that (with attached markers) identify genes of interest or specific segments of DNA B. use restriction endonucleases that serve as 'vectors' (carrying DNA) for introduction of foreign DNA into other cells C. utilize gel electrophoresis that unwinds and cleaves DNA at specific insertion sites D. depend exclusively upon enzymes for separation of DNA strands

A. utilize gene probes that (with attached markers) identify genes of interest or specific segments of DNA

The three main steps in a PCR cycle are _____. A. Denaturation, Annealing, Extension B. Annealing, Primer synthesis, Recombination C. Annealing, Denaturation, Extension D. Polymerization, Chain elongation, Recombination E. Extension, Annealing, Denaturation

A. Denaturation, Annealing, Extension

The single-stranded ends of DNA molecules can be joined together by A) restriction endonucleases. B) DNA ligase. C) DNA polymerase. D) primase. E) helicase.

B) DNA ligase.

In order to clone eukaryotic DNA into prokaryotic cells A) DNA with both exons and introns must be used B) DNA without introns must be added C) RNA with both exons and introns must be used D) exons must be removed from eukaryotic DNA E) introns must be added back to eukaryotic DNA

B) DNA without introns must be added

The purpose of PCR is to A) make more copies of DNA primers to increase protein synthesis B) make many copies of an organism's DNA sequence so a small number of organisms will become large enough to be identified C) make more RNA so large units of protein can be synthesized D) recycle DNA using thermocyclers

B) make many copies of an organism's DNA sequence so a small number of organisms will become large enough to be identified

Human DNA cut with restriction enzyme A can be joined to A) human DNA cut with restriction enzyme B. B) human DNA that is uncut. C) bacterial DNA cut with restriction enzyme A. D) bacterial DNA that is uncut. E) none of the above

C) bacterial DNA cut with restriction enzyme A.

cDNA is A) DNA with both introns and exons that can be cloned into prokaryotes B) DNA with only introns that can be cloned into prokaryotes C) eukaryotic DNA with only exons that can be cloned into prokaryotes D) used to make precursor mRNA

C) eukaryotic DNA with only exons that can be cloned into prokaryotes

The smaller the DNA fragment A) the closer to the origin it will appear B) the brighter color it produces with ethidium bromide C) the faster it migrates during separation by electrophoresis D) the slower it migrates during separation by electrophoresis

C) the faster it migrates during separation by electrophoresis

There are three recent methods of genetic editing. which of the following is NOT a protein zinc finger nucleases TALEN CRISPR/Cas

CRISPR/Cas it is a RNA used to recognize and bing to target DNA

Which of the following could NOT be the recognition site of a restriction endonculease? A) GAATTC CTTAAG B) ATCGAT TAGCTA C) CTGCAG GACGTC D) GCTTGC CGAACG E) GGATCC CCTAGG

D) GCTTGC CGAACG

The purpose of the Southern Blot test is to A) look for a specific nucleotide sequence in the DNA being tested B) to determine how closely two organisms are related C) to identify the size of the fragment that contains the sequence D) a and c E) a, b, and c

D) a and c

all methods of genome editing couple _____ to a DNA recognition sequence- permitting precise cleavage

DNA scissors (nuclease)

restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal

FOK1 nuclease

what happens when the probe hybridizes with an unknown sample of DNA

It labels, tags, or marks, the sequence making it more identifiable the probe is the tagged sequence

What are the characteristics of the recognition sequence?

Nucletoides at one end are complementary to the other end, 2 strands run in opposite directions with same sequence

What recognizes specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides?

Restriction endonucleases

There are three recent methods of genetic editing. which of the following can target specific nucleotides (not triplets) and can chain them together zinc finger nucleases TALEN CRISPR/Cas

TALEN

describe cas9 & clustered repeats (crRNA)

The clustered repeats (crRNA) is from foreign DNA and then they are lined up, make copies, and then when it finds anything like that in the cell, the cas9 cleaves the foreign DNA

In insertional inactivation A) if foreign DNA is inserted, the beta galactosidase marker is inactivated, and the colonies remain white B) if foreign DNA is inserted, the beta galactosidase marker is inactivated, and the colonies turn blue C) if foreign DNA is inserted, the beta galactosidase marker is activated, and the colonies remain white D) if foreign DNA is inserted, the beta galactosidase marker is activated, and the colonies turn blue

a

The lac z gene marker codes for A) b galactosidase, which splits x-gal B) b galactosidase, which makes x-gal resistant to splitting C) ampicillin resistance D) white colonies

a

viral DNA integrated into CRISPR loci of bacterial DNA is known as

acquisition

____ describes any nucleic acid strand with a base sequence that is complementary to the 'sense' or translatable strand

antisense its pretty much the complementary strand for example if the mRNA trans is AUGCGA then the antisense strand is UACGCU

which is more convenient in gene therapy: antisense DNA or antisense RNA

antisense DNA

If host cells are ampicillin sensitive and are plated on a medium containing ampicillin A) only cells that have not taken up the ampicillin resistant vector can grow B) only cells that have taken up the ampicillin resistant vector can grow C) all cells will grow D) ampicillin is inactivated

b

When a dideoxyribonucleotide is added to the tube A) replication of the strand continues B) replication of the strand stops C) replication of the strand is not affected D) replication of the strand is speeded up

b

the first step in cloning a gene is to A) insert a plasmid into a bacterium B) isolate the DNA from the organism that contains the desired gene C) plate cells on agar D) treat plasmids with restriction enzymes

b

In Sanger, or chain termination, sequencing DNA: a. DNA is amplified by means of thermal cycling and specialized enzymes b. the sequence of bases in the DNA can be inferred from the relative lengths of the various DNA molecules that are synthesized from each of the four reaction vessels c. the sequence of DNA bases is determined through electrophoresis of restriction enzyme fragments d. Thousands of different probes are attached to a single small plate in order to identify the primary structure of a long segment of DNA through hybridization

b. the sequence of bases in the DNA can be inferred from the relative lengths of the various DNA molecules that are synthesized from each of the four reaction vessels

what causes blunt or sticky ends

blunt ends are from giametric cut sticky ends are from staggered cuts this happens with the restrictive endonuclease cuts the dna at a specific seuence

When cloning a vector for stem cell research, how is the PCR fragments transformed into competent cells

by the chemical method or electroporation *know: transform into competent cells by the chemical method or by electroporation* slide 47

For DNA amplification to occur, which of the following are needed? A) loose ribonucleotides B) RNA primers C) thermostable DNA polymerase D) b and c E) all of the above

c

Restriction enzymes A) cut donor DNA evenly so smooth edges result B) cut donor DNA but do not affect plasmids C) make staggered cuts at specific sequences in DNA in both donor DNA and plasmid D) are used to incorporate plasmids into bacterial host cells

c

The primer used in Sanger sequencing A) can have any nucleotide sequence B) must have a sequence beginning and ending with the same nucleotide C) has a nucleotide sequence complementary to the 3' end of the region to be copied D) has a nucleotide sequence complementary to the 5' end of the region to be copied

c

what is the value of cDNA

cDNA can be transcribed and translated by the bacterial cell machinery

define cloning vectors expression vectors cosmid vectors & yeast artificial chromosomes (YAC) know

cloning vectors- replicate/multiply DNA segments expression vectors- translate the introduced DNA segment cosmid vectors & yeast artificial chromosomes (YAC) INCREASE payload size for foreign DNA

the sequence obtained by the gel is the ___ strand

complementary strand

crRNA generated from integrated viral DNA is known as

crRNA biogenesis

Plasmids are put into bacterial cells by A) restriction enzymes B) DNA ligase C) binding of cohesive sticky ends D) transformation

d

Taq polymerase starts copying at A) the end of free single-stranded RNA B) any open point C) RNA primers attached to the end of the desired gene D) DNA primers attached to the end of the desired gene

d

To carry out Sanger sequencing a mixture is needed containing A) single-stranded DNA B) DNA polymerase C) four deoxyribonucleotides A, T, C, G D) all of the above

d

how does adding ddNTP result in generation of fragments of different length?

ddNTP terminates the strand synthesis

donor DNA vs vector DNA

donor DNA- contains genes to be transferred vector DNA- vehicle for delivery genes to host cell

in gene therapy, correction of defective genes are done through ex vivo and in vivo therapy explain the two

ex vivo therapy: - tissue cells are removed from patient for introduction (transfection) of recombinant normal gene -altered cells reintroduced by transfusion in vivo therapy - normal DNA introduced directly into patient (via virus)

what is precursor mRNA

exons and introns are transcribed by RNA polymerase creating precursor mRNA

true or false Restriction enzymes cut only at specific sites and therefore are not useful for genetic engineering.

false

true or false Prokaryotic DNA contains both exons and introns.

false Prokaryotic DNA contains only exons. Introns are not present. euk have both exons and introns.

When beta galactosidase cleaves x-gal, white colonies result. A) True B) False

false, When beta galactosidase cleaves x-gal, a blue compound forms and blue colonies result. Recombinant DNA that can not make beta galactosidase results in white colonies.

true or false RNA polymerase removes exons from precursor RNA.

false, RNA polymerase transcribes the whole gene, including both introns and exons. Introns are later removed.

When foreign DNA and plasmid are both cut with the same restriction enzyme and mixed together, all molecules will form recombinants. A) True B) False

false, Some plasmids will reanneal without incorporating foreign DNA.

the DNA probe will bind to identical nucleotide sequences on the test sample. A) True B) False

false, The DNA probe will bind to complementary nucleotide sequences on the test sample

true or false the organism is cloned and multiplied

false, the DNA molecule is cloned and multiplied

When DNA is heated, primers anneal to DNA strands. A) True B) False

false, the strands denature (become single stranded)

___ consists of a short stretch DNA of a known sequence that will base pair with a stretch of DNA that has a complementary sequence

gene probe

_________ implies tweaking of normal cellular metabolism

genetic recombination

RECOMBINANT ORGANISMS PRODUCED THROUGH THE INTRODUCTION OF FOREIGN GENES ARE CALLED

genetically modified organisms (GMO) or transgenic organisms

the systemic study of an oranisms genes and their functions

genomics

____ is an enginered union of crRNA and tracRNA (which is needed to direct cas protein to cleave at the binding site

guide RNA (gRNA)

complementary pairing of nucleic acids from different sources

hybridization

what test utilizes transfer membrane to isolate specific sequences

hybridization test

What does it mean to transform into a competent cell?

if I want to take that plasmid with stem cell capabilities into a cell, the cell has to be compenent. in other words, for a cell to be competent it has to have specialized characteristics. transformation requires that the cell that takes up the DNA has specialized properties. I will achieve transformation by chemical method or electroporation -king

foreign DNA generated from integrated viral DNA

interference

complementary DNA or cDNA are copied from

mRNA tRNA rRNA and other RNA

___ are useful in identifying modified cells and are often genes for abx resistant

marker genes

the study of the complete complement of small chemicals precent in a cell at any given time. provides a snapshot of the physiological state of the cell and the end products of its metabolism

metabolomics

the study of all the genomes in a particular ecological niche, as opposed to individual genomes from single species

metagenomic

construction and transfer of genes between organisms

molecular (genetic) cloning

what are introns?

noncoding section of the RNA transcript that is spliced out before the RNA molecule is translated into a protein

___ sequences of DNA bases that are identical when read 5' to 3' direction on one strand and the opposite direction on the complementary strand

palindromes

why are bacterial cells unable to process eukaryotic mRNA

prok cell dna do not contain introns and prok cells are therefore unable to remove the introns from the euk dna and make functional mRNA. therefore reverse transcriptase has to convert the purified euk mRNA into double stranded DNA (cDNA) which then can be cloned into bacterial cell

the study of an organisms complement of proteins and functions mediated by the proteins

proteomics

the resultant gene and plasmid combination is known as

recombinant

artificially engineered DNA creating unnatural sequence of genes or regulators.

recombinant dna

___ are natural bacterial enzymes that degrade (cleave) foreign DNA

restriction enzymes

differences in the cutting patterns of the restrictive endonuclease give rise to restriction fragments of differing lengths, known as ___

restriction fragment length polymorphisms (RFLP)

the pieces of DNA produced by the restriction endonuclease are termed

restriction fragments

what is the role of reverse transcriptase

reverse transciptase converts the purified euk mRNA into double stranded DNA. the resulting DNA is called the cDNA, which can then be cloned into a bacterial cell

amplicon

template DNA

what can be learned from the pattern on the filter

the radioactive probe will bind to the complementary base of the DNA fragment and then with xray *the position of the radioactive probe can be seen*

altered DNA is introduced to euk cells by ___-

transfection

Non-viral transfer in bacteria and simple euk cells

transformation

DNA fragments can hybridize with plasmids that have been cut by the same restriction enzyme. A) True B) False

true

DNA ligase enzyme joins the cohesive ends of the desired DNA fragment with the cohesive ends of the plasmid. A) True B) False

true

Using PCR, over 1 million copies of DNA can be made from a single strand of DNA overnight. A) True B) False

true

true or false The joining of sticky ends involves the formation of phosphodiester bonds.

true

true or false The position of the bound radioactive probe is made visible on x-ray film.

true

true or false To sequence DNA by this method, the DNA must first be made in single stranded form.

true

true or false Examining the electrophoresis gel and reading from bottom to top, one base at a time, gives the sequence of DNA.

true

What are sticky ends

when the cleaves, the dna strands are not center. so they are staggered and antiparallel. the ends are classed sticky ends. each fragments has a single stranded end a few nucleotides and single stranded end of the two fragments are complementary. these single stranded ends can pair with each other (sticky ends). once the ends have paired, they are rejoined together by DNA ligase to reform the phosphodiester bonds in DNA

three methods of genetic editing

zinc finger nucleases TALEN CRISPR/Cas