Ch 19 Molecular Genetic Analysis

Question 1

molecular techniques for locating, isolating, altering, and studying DNA segments
combines DNA from two distinct sources

Answer 1

Recombinant DNA technology

Question 2

enzymes that recognize specific base sequences in DNA and make double-stranded cuts at those sites

Answer 2

restriction endonucleases (restriction enzymes)

Question 3

where do restriction enzymes come from?

Answer 3

bacteria naturally produce restriction enzymes, which are used in defense against viruses

Question 4

how can a bacterium protect its own DNA from a restriction enzyme

Answer 4

by modifying the recognition sequence, usually by adding methyl groups

Question 5

describe the recognition sequences that restriction enzymes target

Answer 5

usually 4-8 bp long, and are palindromic

Question 6

what occurs when a restriction enzyme makes a staggered cut in the DNA?

Answer 6

cohesive ends (sticky ends) are generated, which are complementary to each other

Question 7

how can any two fragments cleaved by the same enzyme pair?

Answer 7

restriction enzymes pair with and cut specific recognition sequences. any two fragments cleaved by the same enzyme have the same sequence, which will have complementary ends and pair

Question 8

why are shorter recognition sequences more frequent than longer recognition sequences

Answer 8

it’s easier to locate a specific sequence with fewer amount of nucleotides than a sequence with more nucleotides

Question 9

describe how the CRISPR-Cas system serves as an adaptive RNA defense system that remembers and destroys foreign invaders

Answer 9

When foreign DNA enters the cell, it is cut up and inserted into the spacers of the CRISPR array, to serve as a memory.
CRISPR array is transcribed into a long pre-mRNA, which is cleaved into short crRNAs
crRNAs combine with Cas proteins to form CRISPR-Cas complexes
When foreign DNA enters the cell again, these complexes recognize and bind, and the Cas protein cleaves the foreign DNA

Question 10

what does the CRISPR array consist of?

Answer 10

CRISPR array consists of series of palindromic sequences separated by unique spacers.

Question 11

DNA sequences in foreign DNA that match the spacer elements in the CRISPR arrray

Answer 11

protospacers

Question 12

what are the purpose of the Cas proteins in the CRISPR-Cas complex?

Answer 12

Cas proteins have nuclease activity, able to cut DNA
When the CRISPR-Cas complex binds to its complementary sequence in foreign DNA, it cleaves it and makes it nonfunctional

Question 13

how can CRISPR-Cas9 system be used for genome editing?

Answer 13

crRNA and tracrRNA are genetically engineered to form a single guide RNA (sgRNA), which is capable of being altered to direct the CRISPR-Cas9 complex to any DNA sequence desired

Question 14

what is a protospacer-adjacent motif (PAM) and why is it required in the target DNA sequence of the CRISPR-Cas9 system?

Answer 14

a PAM is a short sequence (5’-NGG-‘3), that occur at random places through the genome
the CRISPR-Cas9 complex must associate with the PAM.

Question 14

describe the CRISPR-Cas9 system and how it edits the genome

Answer 14

sgRNA and Cas9 protein combine to form an effector complex
CRISPR-Cas9 effector complex associates with PAM, which allows the Cas9 to unwind the DNA. sgRNA pairs with it complementary sequence, and then Cas9 cleaves the protein
the DNA can be repaired by nonhomologous end joining or homology directed repair

Question 15

describe nonhomologous end joining repair

Answer 15

DNA ends are joined without using any template, producing small insertions and deletions at the break site, leading to frameshift mutations that disrupt the coding sequence and disable the gene

Question 16

describe how homology directed repair is used to edit a DNA target sequence
why is this not efficient?

Answer 16

a donor piece of DNA is provided used as a DNA template to repair the DNA break
not highly efficient, as often the DNA ends are connected without the donor DNA being inserted

Question 17

list the advantages of CRISPR-Cas9

Answer 17

-long sgRNA nucleotide sequence allows researchers to produce unique cuts within genomic DNA; alteration of sgRNA allows for almost any gene to be edited
-can be used in intact cells
-can be used in many different species
-can be used for gene therapy to correct genetic defects and treat viral infectious diseases
-used for genetic modification of crops and animals

Question 18

list the limitations of CRISPR-Cas9

Answer 18

-mismatches are often, which result in Cas9 cleaving the wrong target DNA
-also hard to predict where or when cleavage occurs
-creates genetic mosaics, in which DNA is edited in some cells and not in others
-difficult to get Cas9 components into the cell

Question 19

explain how gel electrophoresis is performed

Answer 19

-small wells are made in one end of the gel, where solutions of DNA fragments are placed
-an electrical current is passed through the gel
-DNA fragments move toward the positive pole
-small fragments move farther than larger fragments
-a dye specific for nucleic acids added, so DNA fragments appear as bands
-DNA fragments of a known size (placed in one of the wells) is used to compare the unknown fragments to determine the sizes

Question 20

in gel electrophoresis, what charge do the DNA fragments move to?

Answer 20

positive charge

Question 21

in gel electrophoresis, what fragments move farther?

Answer 21

smaller fragments move farther than larger fragments

Question 22

how can researchers locate the desired fragments in a large pool of DNA

Answer 22

using a probe

Question 23

what is a probe?

Answer 23

a DNA or RNA molecule with a base sequence complementary to a sequence in the gene of interest, which can be used to locate a specific gene or DNA sequence

Question 24

what are the three different techniques for transferring denatured fragments to a permanent solid medium, and how do they differ?

Answer 24

Southern Blotting - denatured single stranded DNA fragments transferred
Northern Blotting - denatured RNA transferred
Western Blotting - proteins transferred

Question 25

describe how DNA fragments are located with a probe

Answer 25

1) DNA fragments are cut up by restriction enzymes and separated by gel electrophoresis
2) Separated fragments are denatured and transferred to a permanent solid medium (nitrocellulose or nylon membrane) by Southern Blotting
3) Membrane is placed in a hybridization solution containing a labeled probe
4) The probe binds to any fragments that are complementary to it

Question 26

what is the purpose of Polymerase Chain Reaction (PCR)?

Answer 26

PCR allows DNA fragments to be amplified quickly

Question 27

list the required materials for polymerase chain reaction (PCR)

Answer 27

target DNA
DNA polymerase (Taq polymerase)
primers that have a 3’-OH group to add nucleotides to
all four deoxyribonucleoside triphosphate (dNTPs)
salts

Question 28

describe the three steps of polymerase chain reaction (PCR)

Answer 28

1) denaturation - DNA is heated to 90º-100ºC, which breaks the hydrogen bonds and separates the two strands
2) annealing - DNA solution is quickly cooled to 30º-60ºC to allow primers to attach to their complementary sequences on the template strands
3) extension/elongation - the solution is heated to 72ºC, which allows DNA polymerase to synthesize new DNA strands, producing two double-stranded DNA molecules

Question 29

why is the use of Taq polymerase important for PCR?

Answer 29

Taq polymerase is stable at high temperatures, therefore, it doesn’t denature in the first step of PCR, when the solution is heated, and can continue throughout the rest of the cycle

Question 30

what is gene cloning?

Answer 30

producing identical copies (clones) of an original piece of DNA within a bacterial cell

Question 31

how does gene cloning work?

Answer 31

a DNA fragment is inserted into a bacterial cell (host) and the cell replicates the DNA
each time the cell divides, the copies of the DNA are passed to each daughter cell to produce genetically identical cells
cells are then lysed to release their DNA, and the desired fragment is isolated

Question 32

a stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell; used for gene cloning

Answer 32

cloning vector

Question 33

what are three important characteristics of a cloning vector, and why are they significant?

Answer 33

1) origin of replication - so the vector can replicate within the cell
2) selectable markers - enable cells containing the vector to be identified
3) one or more restriction sites - where DNA fragment can be inserted into

Question 34

how are plasmids used for gene cloning?

Answer 34

plasmids are commonly genetically engineered and used as cloning vectors. they have origin of replications, therefore can self-replicate; multiple restriction sites and selectable markers

Question 35

describe the process of inserting a DNA sequence into a plasmid vector

Answer 35

1) plasmid and foreign DNA (containing fragment of interest) are both cut by the same restriction enzyme, producing complementary sticky ends
2) when mixed together, the sticky ends pair, and the foreign DNA and plasmid are joined
3) DNA ligase seals the nicks

Question 36

what are linkers and how are they used in the event that restriction sites are not available at a place where DNA needs to be cut?

Answer 36

linkers are small synthetic DNA fragments that have restriction sites, and they attach to the ends of the DNA. restriction enzymes can then cut at the restriction sites of the linkers to generate sticky ends that then pair with the plasmid

Question 37

what process allows a plasmid vector (containing the DNA fragment of interest) to be introduced into bacterial cells

Answer 37

transformation - bacterial cells take up DNA from external environment

Question 38

what genes are commonly used as selectable markers in plasmids?

Answer 38

genes that have antibiotic resistance

Question 39

explain how the lacZ gene can be used to screen for bacteria containing plasmids

Answer 39

-Use a plasmid containing fragment (front end) of lacZ gene
-Bacteria with lacZ- are missing the fragment, and are transformed by the plasmid
-If no foreign DNA is inserted into the lacZ gene of the plasmid, bacteria have original (nonrecombinant) plasmid and produce ß-galactosidase
-If foreign DNA is successfully inserted into one of the restriction sites of the lacZ gene of the plasmid, it disrupts the gene and ß-galactosidase is not produced
-The colors of bacterial colonies are used to determine if a recombinant or intact plasmid is present (blue = intact; white = recombinant)

Question 40

why can cells only with ampicillin (antibiotic resistance gene) grow on the plates?

Answer 40

The cells that are successfully transformed by plasmids have the ampicilliin-resistance gene, allowing them to survive and grow. Other cells that aren’t transformed die.

Question 41

what color do the bacteria change if foreign DNA has been successfully inserted into the plasmid? if foreign DNA isn’t? why?

Answer 41

if foreign DNA has been successfully inserted, the bacterial colonies remain white, due to them not being able to produce ß-galactosidase.
if foreign DNA isn’t inserted, the bacterial colonies turn blue, as they are able to produce ß-galactosidase

Question 42

what is an expression vector, and how does it differ from a cloning vector?

Answer 42

an expression vector includes all the components a cloning vector has (origin of replication, selectable markers, restriction sites), along with sequences required for transcription and translation, in order to produce the protein the gene encodes

Question 43

collection of clones containing all DNA fragments for one source

Answer 43

DNA library

Question 44

collection of bacterial colonies or phages with DNA fragments of entire genome of an organism

Answer 44

Genomic library

Question 45

collection of clones with DNA fragments that transcribe into mRNA

Answer 45

cDNA library

Question 46

probes can be used to screen DNA libraries. how is a probe obtained when the gene of interest has not yet been isolated?

Answer 46

A similar gene from another organism can be used as the probe, as it may have a related sequence that can hybridize with the target DNA

Question 47

explain how synthetic probes are created to screen DNA libraries when the protein encoded by the gene is known

Answer 47

the amino acid sequence of the protein is determined, and possible nucleotide sequences are deduced from the amino acid sequence. then, a mixture of all the possible nucleotide sequences is used as a probe

Question 48

technique used to determine the chromosomal location of a gene or specific DNA fragment

Answer 48

in situ hybridization

Question 49

describe how in situ hybridization (with fluorescence) works

Answer 49

-cells are fixed and chromosomes are spread on a microscope slide
-DNA/RNA is denatured
-a labeled probe is applied to the slide. carrying fluorescent dyes that can be seen directly through the microscope

Question 50

what are dideoxyribonucleoside triphosphate (ddNTPs) and why are they essential for dideoxy sequencing

Answer 50

ddNTPs are identical to dNTPs, except they lack a 3’-OH group. when added incorporated into a synthesizing DNA strand, no more nucleotides can be added, terminating DNA synthesis

Question 51

what materials are added to each test tube in dideoxy sequencing?

Answer 51

-target DNA
-primers
-all four types of dNTPS
-one type of ddNTP (each tube receives a different ddNTP)
-DNA polymerase

Question 52

describe the process of dideoxy sequencing

Answer 52

-DNA solution is split into four test tubes, containing primers, all four dNTPs, DNA polymerase, and one of the four types of ddNTPs in each tube
-Nucleotides are added to the 3’ end of the primer, using target DNA as the template
-a ddNTP incorporated into the chain terminates DNA synthesis
-synthesis terminates at different position on different strands, generating DNA fragments with different lengths
-fragments are separated by gel electrophoresis, and the sequence is read

Question 53

in dideoxy sequencing, how is the original DNA template determined?

Answer 53

after the fragments are separated in gel electrophoresis, the sequence is determined starting from the bottom. the sequence complementary to this sequence is the original DNA

Question 54

why do you read the sequence starting at the bottom of the gel electrophoresis in dideoxy sequencing?

Answer 54

the smallest fragments travel the furthest, so the nucleotides at the beginning of the synthesized strand are at the bottom

Question 55

the use of DNA sequences to identify individual people

Answer 55

DNA fingerprinting (DNA profiling)

Question 56

very short DNA sequences repeated in tandem; used for DNA fingerprinting

Answer 56

microsatellites (short tandem repeats)

Question 57

why are microsatellites used for most DNA fingerprinting?

Answer 57

they are found at many loci throughout the genome, and people vary in the number of copies of microsatellites

Question 58

study of gene function that begins with mutant phenotype and proceeds to a gene that encodes the phenotype

Answer 58

forward genetics

Question 59

study of gene function that begins with a DNA sequence and proceeds to the phenotype by altering the sequence or by inhibiting its expression

Answer 59

reverse genetics