Chapter 19 - Recombinant DNA Technology

Question 1

The first recombinant DNA experiments were done in the __s. It combined DNA from __ sources to create a __ DNA molecule.

Answer 1

70s.

different; novel/new

Question 2

__ means DNA from different sources is combined to create a novel DNA molecule. Also known as genetic __.

Answer 2

Recombinant.

Engineering

Question 3

Prior to recombinant tech, the genotype had to be __ from the __. Mutations could only be created __; and because of their complexity, __ organisms could not be studied with the old techniques.

Answer 3

inferred.

randomly; eukaryotic

Question 4

One of the challenges of studying genetics at the molecular level (as opposed to __ the genotype) is finding the gene of interest is like finding a __ __ __ __. Once found, producing enough of the gene and gene __ would be an enormous undertaking.

Answer 4

inferring; needle in a haystack.

product

Question 5

A fundamental requirement for DNA manipulation at the molecular level is __ and __ DNA fragments. This is made possible by __ enzymes called __ __ (aka __ __).

Answer 5

cutting and joining.

bacterial; restriction endonucleases; restriction enzymes.

Question 6

The biological purpose of restriction enzymes is to make __-__ cuts in DNA of viruses and causing the foreign DNA to __.

The bacterial DNA isn’t targeted because it is “__” by a specific pattern of ___.

Answer 6

double-stranded; degrade.

“marked”; methylation

Question 7

Brake down the nomenclature of the following __ enzyme:

EcoR1

Answer 7

Restriction;
Eco = E. coli (source);
R1 = 1st restriction endonuclease/enzyme discovered.

Question 8

Most __ enzymes are able to recognize small sequences about 4 - 6bp in length. The recognition sequence is always a __ (read the same way __ and __). They can be bisected __ and __.

Answer 8

restriction.

palindrome; forward; backward.

horizontally; vertically

Question 9

Complete the following recognition sequence for restriction enzyme XyzR2: 3’ TTC

a) 5’ GAA
b) 5’ AAG
c) GAA 5’
d) more than one of the above

Answer 9

d) more than one of the above

b) 5’ AAG AND c) GAA 5’

Question 10

What are the 2 types of cuts made by restriction enzymes? What are the ends of one of them called, and why does it have that name?

Answer 10

(1) Blunt end cuts (no single-stranded ends)
(2) Staggered cuts (single-stranded ends).

Staggered cuts make “sticky” ends or “cohesive” ends. They’re so named because the ends are complimentary and will ‘stick’ or reanneal if given the opportunity.

Question 11

Blunt end vs. sticky end cuts: which is more advantageous?

Answer 11

blunt

Question 12

DNA from different sources can be __ by the same __ enzyme to produce complimentary __.

Answer 12

digested; restriction; termini

Question 13

PCR is DNA __ converted into a test-tube reaction. A fragment can be __ over 1B fold in one hour. It is the most __ method for DNA analysis. It can analyze DNA from a single __.

Answer 13

cloning.
amplified.
sensitive.
cell.

Question 14

PCR is a chain reaction because it __ at every step.

Answer 14

doubles.

Question 15

True PCR product is produced at the __ round of replication. But in general, after 30 cycles, DNA is amplified ^, or more than one billion fold.

Answer 15

3rd.

2^30

Question 16

To order PCR primer, one needs to know the sequence of the __ of the target element.

Once ordered, DNA is __, primer is added, primer attaches to the __ of each strand, and new DNA is synthesized. Rinse and repeat.

Answer 16

termini.

denatured; termini

Question 17

One of the problems to overcome in automating PCR was finding a __-__ DNA polymerase. The first used was __ from a thermophilic prokaryote, but it is an __-__ polymerase because it lacks __ activity. Subsequent polymerases with better __ have been since been discovered.

Answer 17

heat-stable.
Taq; error-prone; proofreading.
accuracy

Question 18

What can real-time PCR do that automated PCR can’t do? Why is this valuable (i.e. what can it be used to test)?

Answer 18

Real-time PCR can quantitate the amount of PCR product.

It’s valuable because it can “measure” changes in gene expression - if you add mRNA from regular cells vs. cancer cells, the amount of PCR product is directly proportional to the amount of mRNA, so you can infer that the cancer cells are expressing more mRNA than normal cells.

Question 19

Gene discovery is becoming more the result of “__ __” of extensive and rapidly growing DNA databases (the latin phrase “In __” has been coined). The field is called ___, but there are many other names like genomics, proteomes, transcriptomes, etc.

Answer 19

“data mining”; “In Silico”.

bioinformatics.

Question 20

__ __ __ is a technique that uses a complimentary “probe” to determine the cellular or chromosomal location of the __ or __ product. Basically, it is in vivo ___ and __ of a biomolecule.

Answer 20

In situ hybridization; gene; gene product.

tracking and visualization

Question 21

DNA __ exploits the highly-variable sequences among different individuals. These __ sequences do NOT have to be a gene; many are __ or __ __ __. The sequences of interest are amplified using __.

Answer 21

fingerprinting.
polymorphic; microsattelites; short tandem repeats (STRs).

PCR

Question 22

The 1st method used for DNA fingerprinting was __ __ __ __ (RFLP). It uses __ enzymes that target small sequences (called __ sequences) along the length of the molecule. The __ enzyme “digests” each __ sequence creating DNA __ of varying ___. People vary in the number of __ sequences, so they will yield fragments that vary in __ and __. These fragments will separate according to their __ during electrophoresis.

Answer 22

restriction fragment length polymorphism.
restriction; recognition.
restriction; recognition; fragments; lengths.
recognition, length; number.
size.

Question 23

A RFLP is used to test 3 individuals. One person has banding found in pattern D; the second had banding found in pattern Q; and the third person has banding found in both patterns D and Q. what can you conclude about these individuals?

Answer 23

Person 1 is homozygous for pattern D; person 2 is homozygous for pattern Q; and person 3 is heterozygous for patterns D and Q.

Question 24

Using __ or __ __ __ for DNA fingerprinting is more advantageous than using RFLPs because there are more “__” to work with.

Answer 24

microsattelites; short tandem repeats; ‘alleles’

Question 25

DNA __ determines the precise nucleotide order in a DNA fragment. It is a fully __ process, and it is far cheaper to __ __ __.

Answer 25

sequencing.

automated

send it out

Question 26

Germline transformation means that the genetic manipulation performed on __ animals is ___ (the progeny carry the __ and pass it to their offspring).

Answer 26

transgenic; heritable; transgene.

Question 27

Gene silencing uses __ to “__ down” gene expression. A __ is needed to get the cell to express the __-__ RNA molecule. The cell will then convert the __-__ RNA to __ by using __.

Answer 27

siRNAs; ‘knock’.
vector; double-stranded.
double-stranded; siRNA; Dicer.

Question 28

__/__ genome editing selectively deletes (“__-out”) or inserts (“__-in”) a desired DNA sequence at a __ chromosomal sequence. It’s the most efficient means of inducing ___.

Answer 28

CRISPR/Cas9; “knocks-out”; “knocks-in”; specific.

mutations.

Question 29

Cas9 is an __ that cuts the genomic DNA. Specificity of the cut is mediated by a __ RNA (-RNA; it effectively directs Cas9 to the specific site). Cas9 then makes a __-__ cut, and the changes/mutations are created when the cell attempts to __ the damage.

Answer 29

endonuclease.
guide; gRNA.
double-stranded; repair.

Question 30

The fastest and cheapest genome editing tool is?

Answer 30

CRISPER/Cas9

Question 31

If the sequence 5’ AAGC 3’ were part of recognition sequence for a restriction endonuclease, what would be the *next* four nucleotides of the same strand?
A. 5’ GATT 3’ 
B. 5’ GCTT 3’ 
C. 5’ CTAA 3’ 
D. 5’ TACG 3’ 
E. 5’ AAGC 3’

Answer 31

B. 5’ GCTT 3’

Question 32

Restriction fragment length polymorphisms (RFLP) can be used to study the inheritance of human genetic diseases by
A. Distinguishing the regions of the chromosomes that contain genes from the regions that do not contain genes.
B. Following the co-inheritance of the gene for the disease with point mutations that alter the recognition sequence of a specific restriction endonuclease.
C. Analyzing the co-inheritance of the gene for the disease with other phenotypic markers.
D. Promoting increased levels of genetic recombination during meiosis.
E. Determining whether the genetic mutation responsible for the disease is dominant or recessive.

Answer 32

B. Following the co-inheritance of the gene for the disease with point mutations that alter the recognition sequence of a specific restriction endonuclease.

Look at the answers themselves!!!! You don’t use phenotype with RFLPs; but RFLPs DO target recognition sequences, and they’re specific to each restriction enzyme.

Question 33

Which of the following would not be a practical application of recombinant DNA technology?
A. Production of a specific RNA or protein in large quantities.
B. Creation of transgenic organisms or other alterations to its genotype.
C. Diagnostic testing in medical diseases
D. Gene therapy — correction of genetic diseases
E. Select this answer if all of the above choices describe practical applications of recombinant DNA technology.

Answer 33

E. Select this answer if all of the above choices describe practical applications of recombinant DNA technology.