Chapter 19

Question 1

Recombinant DNA Technology

Answer 1

Set of molecular techniques for locating, isolating, altering, combining, and studying DNA segments; also commonly called genetic engineering

Question 2

Restriction Enzymes (or Restriction Endonuclease)

Answer 2

Enzymes that recognize particular base sequences in DNA and make double stranded cuts (restriction sites) nearby

Question 3

Gel electrophoresis

Answer 3

Technique for separating charged molecules (such as proteins or nucleic acids) into fragment sizes

Question 4

Ethidium Bromide

Answer 4

It has a role as a DNA intercalculator, inserting itself inbetween the base pairs in the double helix (don’t have to remember this part rn)

• basically works with gel electrophoresis under UV light to reveal fluorescent bands that shows the various DNA bands separated by size

Question 5

PCR

Answer 5

PCR (polymerase chain reaction)

a laboratory nucleic acid amplification technique used to denature short segments of DNA or RNA sequences using DNA polymerase

Question 6

Taq DNA Polymerase

Answer 6

DNA polymerase commonly used in PCR reactions due to its thermostability. Isolated from the bacterium thermus aquaticus, the enzyme is stable at high temperatures so it is not denatured during the strand-separation step of the cycle

Question 7

Melting temperature

Answer 7

The temperature at which half of the DNA duplexes denatures

Question 8

DNA fingerprinting

Answer 8

used in forensic testing or paternity testing, uses PCR to amplify micro satellite

Question 9

Real-Time PCR

Answer 9

real-time PCR (qPCR) modification of PCR used to measure starting nucleic acids; DNA is simplified and measured as reaction proceeds

Question 10

Gene-Cloning

Answer 10

insertion of DNA fragments into bacteria in such a way that the fragments will be stable and copied by bacteria (ex. E. coli)

Question 11

Cloning vector

Answer 11

Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for transfer to a host cell

Question 12

DNA ligase

Answer 12

Enzyme that catalyzes the formation of a phosphodiester bond between adjacent 3’ - OH and 5’ phosphate groups in a DNA molecule

Question 13

competent E. Coli

Answer 13

The ability of E. coli to take up DNA from the environment (to be transformed)

Question 14

transformation

Answer 14

mechanism by which DNA found in the environment is taken up by a cell. after transformation recombination may take place between the introduced genes and the cellular chromosomes

Question 15

CRISPR Cas genome editing

Answer 15

A method of splicing DNA to insert a donor, DNA CAS protein is expressed from streptococcus which uses S9RNA that has 20 nucleotides that are complimentary to target DNA. PAM is recognized by every CRISPR CAS 9 and occurs every 8bp

Question 16

microsatellite DNA (STRs)

Answer 16

Short tandem repeat sequences found at many loci in a human genome

Question 17

Sanger (dideoxy) sequencing

Answer 17

a technique for DNA sequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication.

Question 18

4 Key Innovations of Molecular Genetics since discovery of three-dimensional DNA structure by Watson, Crick, Franklin and Wilkins:

Answer 18

1. Development of recombinant DNA technology
2. The invention of the polymerase chain reaction
3. Development of quick methods to determining DNA sequences
4. Engineering of CRISPR Cas System

Question 19

Biotechnology

Answer 19

Use of biological processes, particularly molecular genetics and recombinant DNA technology to produce products of commercial value

Question 20

Molecular Genetics

Answer 20

techniques used to study biology at the level of DNA, proteins, and other molecules; recombinant DNA technology is an important component of molecular genetics

Question 21

Most recognition sequences (or restriction sites) are how many bp long?

Answer 21

4-8 bp and are “palindromic” that read the same 5’ to 3’ on the two complementary DNA strands

Question 22

Overhanging sticky or cohesive ends

Answer 22

complementary fragments that can spontaneously pair to rejoin DNA fragments that have been cut with the same restriction enzymes.

can have either 5’ or 3’ strand overhanging ends.

Question 23

HindIII Example Cohesive Cut

Answer 23

5’-A|AGCTT- 3’

3’-TTCGA|A- 5’

HindIII cuts the sugar-phosphate backbone of each strand at the point indicated by the line, generating in this example fragments with short, single-stranded overhanging 5’ ends or cohesive ends:

5’-A AGCTT- 3’

3’-TTCGA A- 5’

Question 24

Blunt Fragments Example

Answer 24

PvuII cuts in the middle of its recognition sequence, and the cuts on the two strands are directly opposite each other, producing blunt-ended fragments.

5’-CAG|CTG-3’

3’-GTC|GAC-5’

5’-CAG CTG-3’

3’-GTC GAC-5’

Question 25

In gel electrophoresis DNA migrates towards the positive pole because….

Answer 25

Because DNA has a strong negative charge due to it’s phosphate groups

Question 26

How does gel electrophoresis separate DNA by size?

Answer 26

Gel hinders DNA mobility, smaller DNA fragments will migrate faster because their mobility will be less hindered than larger fragments

Question 27

What is needed for two cohesive fragments to be ligated together?

Answer 27

Both need the same 3’ or 5’ overhanging ends

The single stranded parts need to be complementary

size of overhang has to be the same

Question 28

Can two blunt ends DNA fragments be ligated (idk if ligated is a word but whatever) together? why?

Answer 28

Yes, it would need two enzymes that leave blunt ends and there is no need for complementary ends (matching sequences)

Question 29

What are the two ways to amplify DNA?

Answer 29

PCR (polymerase chain reaction)

and

Gene cloning (inserting gene/DNA segment into plasmid, introducing E. coli, E. coli then replicates)

Question 30

PCR Cycle Steps

Answer 30

1. Denaturing 94C: requires high temp, close to boiling/strands separate
2. Annealing 55C: primer (single strand short DNA) anneals to base pair with complementary sequence
3. Extension 72C: polymerase can now extend/add nucleotides to 3’ hydroxyl and synthesize DNA

Each cycle number of DNA molecules double

(note: don’t need to memorize temps but given for example but like idk do whatever u want just know that denaturing is always the highest)

Question 31

1kb is equal to how many bp?

Answer 31

1000bp (and 500bp would be like 0.5kb)

Question 32

Melting temperature is higher of a primer during PCR when?

Answer 32

tm influenced by: length of primer and *GC (base pair) content

*GC pairs have more hydrogen bonding - when there is more hydrogen bonding between the primer and template DNA tm is higher

Question 33

Gene cloning steps

Answer 33

1. Digest - gene of interest is cut using restriction enzymes and plasmid is digested. plasmid is then cut using same restriction enzyme for complementary sticky ends
2. Ligate - cut and glue together vector with DNA ligase
3. Transform - ligation mixture goes into competent E. coli
4. Select - E. coli plasmid contains DNA of interest and will grow and replicate

Question 34

3 important features of a cloning vector

Answer 34

an origin of replication where DNA replication starts

DNA restriction sites

selectable marker (so e.coli cells can be distinguished between with and without plasmid

Question 35

How does the Dideoxy Sequencing (Sanger method) result in DNA strands with different lengths?

Answer 35

Replication is terminated when a specific base is encountered resulting in DNA strands of different lengths

Question 36

3 considerations for performing PCR

Answer 36

melting temperature - consider melting temp when choosing primer

high error rate - taq does not have proofreading 3’ to 5’ exonuclease

extremely sensitive - contamination could be an issue

Question 37

Concept Q: DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length are separated by gel electrophoresis. Which fragment will migrate farthest in the gel? Why?

Answer 37

500bp because smaller fragments migrate faster through gel

Question 38

What is needed to perform PCR?

Answer 38

DNA Template
DNA Primer
dNTPs
Buffer
Thermal cycler