Molecular Genetic Analysis and Biotechnology (Lec 15)

Question 1

Techniques used for locating, altering, and studying DNA segments are considered why type of technology?

Answer 1

Recombinant DNA technology (genetic engineering)

Question 2

The molecular genetics revolution refers to what?

Answer 2

biotechnology: the use of these techniques to develop new products

Question 3

Describe the molecular techniques and their respective order used to isolate, recombine, and amplify genes

Answer 3

isolate DNA segment or gene from remaining DNA; cutting and joining DNA fragments - restriction enzymes; viewing DNA fragments; locating DNA fragments with southern blotting and probes

Question 4

How doe restriction enzymes work?

Answer 4

recognize and cut DNA at specific sequences

Question 5

How does a restriction enzyme protect itself form being digested by its own enzyme in bacteria?

Answer 5

by adding methyl groups to the recognition sequence

Question 6

In restriction enzymes, fragments with short, single-stranded overhanging ends are called what?

Answer 6

cohesive ends

Question 7

In restriction enzymes, even length ends from both single strands are called what?

Answer 7

blunt ends

Question 8

What are the two types of ways to view DNA fragments?

Answer 8

gel electrophoresis; autoradiography

Question 9

True or false?
a probe is DNA or RNA with a base sequence complementary to a sequence in the gene of interest and allow one to locate DNA fragments

Answer 9

true

Question 10

Match the blank with DNA, RNA, or protein
Southern blot = ___
Northern blot = ____
Western blot = ____

Answer 10

Southern blot = DNA
Northern blot = RNA
Western blot = protein

Question 11

How are DNA fragments amplified?

Answer 11

pcr

Question 12

define gene cloning

Answer 12

amplifying a specific piece of DNA via a bacteria cell

Question 13

define cloning vector

Answer 13

a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell

Question 14

What are three things a cloning vector needs?

Answer 14

origin of replication; selectable markers; single cleavage site

Question 15

Circular DNA molecules from bacteria are called what?

Answer 15

plasmids

Question 16

Synthetic DNA fragments containing restriction sites are called what?

Answer 16

linkers

Question 17

What are selectable markers used for?

Answer 17

to confirm whether the cells have been transformed or not

Question 18

Expression vectors contain operon sequences that allow what?

Answer 18

DNA to be transcribed and translated

Question 19

What does the Bt gene encode for in plants?

Answer 19

an endotoxin that inserts into the insect gut cell membrane, paralyzing the digestive tract and forming a pore. The insect stops eating and starves to death

Question 20

What is Taq polymerase?

Answer 20

stable DNA polymerase at high temperature

Question 21

What does real-time pcr show?

Answer 21

quantitatively determines the amount of DNA amplified as the reaction proceeds

Question 22

List some of the limitations of pcr

Answer 22

requires prior knowledge of at least part of the sequence of the target DNA to allow for the construction of the primers; the capacity of pcr to amplify extremely small amounts of DNA makes contamination a significant problem; taq polymerase does not have the capacity to be proofread; the size of the fragment that can be amplified by taq polymerase usually less than 2000 bp

Question 23

List some of the uses of pcr

Answer 23

detecting presence of viruses in blood samples; identify genetic variation; isolate DNA from ancient sources; amplify small amounts of DNA from crime scenes; introduce new sequences into a fragment of DNA

Question 24

a collection of clones containing all the DNA fragments from one source is called what?

Answer 24

DNA library

Question 25

What are cDNA libraries?

Answer 25

consist only of those DNA sequences that are transcribed into RNA

Question 26

True or false? When creating a gene library, some clones contain the entire gene of interest, others include part of the gene, and most contain none of the gene of interest.

Answer 26

true

Question 27

How are DNA libraries screened?

Answer 27

clones of library are plated and then probed

Question 28

The following describe what type of hybridization: | DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell; FISH

Answer 28

Situ Hybridization

Question 29

Isolating genes on the basis of their position on a genetic map, chromosome walking, and chromosome jumping are all aspects of what type of cloning?

Answer 29

positional cloning

Question 30

By making probes complementary to areas of ___ between cloned fragments in a genomic library, we can connect a gene of interest to a previously mapped, linked gene

Answer 30

overlap

Question 31

True or false? | cystic fibrosis is the most common fatal genetic disease in the united states today

Answer 31

true

Question 32

CF is autosomal dominant or autosomal recessive disease?

Answer 32

autosomal recessive

Question 33

What is the incidence rate of CF?

Answer 33

1 in 3,300

Question 34

What is CF characterized by?

Answer 34

chronic lung infections, insufficient pancreatic enzyme production, and increased salt concentration in sweat

Question 35

The CF transmembrane regulator gene encodes for what?

Answer 35

an ion channel necessary to maintain fluid balance

Question 36

Some DNA fragments have different restriction sites due to mutation for the same restriction enzyme; this causes polymorphisms within a population called what?

Answer 36

restriction fragment length polymorphisms

Question 37

ddNTP lacks a 3' OH group, which terminates DNA synthesis. What is ddNTP used in?

Answer 37

Sanger's sequencing method

Question 38

How does sanger sequencing with p32 and gel electrophoresis work?

Answer 38

synthesis terminates at different positions on different strands, which generates a set of DNA fragments of various lengths, each ending in a dideoxynucleotide with the same base; the fragments produced in each reaction are separated by gel electrophoresis; the sequence can be read directly from the bands that appear on the autoradiograph of the gel; the sequence obtained is the complement of the original template strand

Question 39

How does sanger sequencing with fluorescent labels and capillary electrophoresis work?

Answer 39

each of the four ddNTP's is tagged with a different fluorescent dye; fragments that end in the same base have the same colored dye; products are denatured and the fragments produced by the four reactions are mixed and loaded into a single well on a gel; fluorescent dye on the DNA is detected by laser beam; each fragment appears as a peak on the computer

Question 40

Pyrosequencing is a next-generation sequencing technique. Describe it.

Answer 40

DNA is broken up into fragments; adapters containing primer sequences are attached to each fragment, the DNA is made single stranded; each DNA fragment is attached to a bead and surrounded by a drop of solution containing reagents of PCR; the DNA fragment is amplified by PCR; DNA is made single stranded, each bead is forced into a well and layered with sequencing reagents; DNA synthesis takes place within each well; a solution containing a specific dATP flows on the plate; when a nucleotide is added to the growing chain, PPi is released and produces a light-emitting chemical reaction; an instrument records the light emitted by each well; the amount of light is proportional to the number of nucleotides added

Question 41

The following describe what: microsatellites: STRs variable number of copies of repeat sequences possessed by many organisms; detected by pcr; fragments represented as peaks on a graph

Answer 41

DNA fingerprinting (DNA profiling)

Question 42

In regards to DNA fingerprinting and the fragments represented as peaks on a graph, explain the difference between homozygotes and heterozygotes

Answer 42

homozygotes: single tall peak heterozygotes: two shorter peaks

Question 43

Microsatellites, also known as ___ can use pcr primers flanking them to amplify and electrophoresis to identify size variants within and between individuals; each individual as a unique compliment of microsatellietes = a ___ ___

Answer 43

STRs; DNA fingerprint

Question 44

This type of genetics begins with a phenotype to a gene that encodes the phenotype

Answer 44

forward genetics

Question 45

This type of genetics begins with a gene of unknown function, first inducing mutations and then checking the effect of the mutation on the phenotype

Answer 45

reverse genetics

Question 46

In oligonucleotide-directed mutagenesis, the oligonucleotide serves as what?

Answer 46

a primer for | DNA synthesis which produces a molecule with a single mismatched pair

Question 47

Define transgenic animal

Answer 47

an organism permanently altered by the addition of a DNA sequence to its geneome

Question 48

A mouse that carries an inserted DNA sequence at specific locations is called what?

Answer 48

knock-in mice

Question 49

In a knock-out experiment, the phenotype of the knockout mice reveals what?

Answer 49

the function of the gene

Question 50

What do siRNAs do?

Answer 50

silence genes through a process called RNA interference

Question 51

True or false? RNAi can be used for the treatment of human disease. It has shown to reduce the levels of ApoB and blood cholesterol in nonhuman primates

Answer 51

true

Question 52

What are some examples of pharmaceutical products that harness the power of molecular genetics?

Answer 52

human growth factor, insulin, clotting factors

Question 53

What are some examples of specialized bacteria that harness the power of molecular genetics?

Answer 53

vitamin B2, C; various amino acids; thickener; xanthan

Question 54

What are some examples of agriculture products that harness the power of molecular genetics?

Answer 54

round up ready corn, soybeans, wheat

Question 55

What are some examples of genetic testing that harness the power of molecular genetics?

Answer 55

genomic medicine, BRCA 1/2 for therapy decisions

Question 56

What are some examples of gene therapy that harness the power of molecular genetics?

Answer 56

Liver-directed lentirviral gene therapy in a dog model of hemophilia B

Question 57

A geneticist is interested in the immune function of mice and induces random mutations in a number of genes in mice and then determines which of the resulting mutant mice have impaired immune function. This is an example of what?

Answer 57

reverse genetics