Chapter 5: Exploring Genes and Genomes

Question 1

Acts as precise scissors for cutting specific DNA sequences

Answer 1

Restriction enzymes

Question 2

Allows the separation and identification of specific proteins and nucleic acids using gel electrophoresis

Answer 2

Blotting techniques

Question 3

The genome sequences of entire organisms can be determined

Answer 3

DNA sequencing

Question 4

Specific sequences of nucleic acids can be synthesized and used to identify or amplify other nucleic acids

Answer 4

Solid-phase synthesis of nucleic acids

Question 5

Allows the billion- fold amplification of DNA to obtain sufficient quantities for further characterization

Answer 5

Polymerase chain reaction

Question 6

Identifying unknown restriction fragments on a gel?

Identifiying an DNA sequence on a gel, using a phosphate labeled probe.

Answer 6

Southern blotting

Question 7

Identifying an RNA sequence on a gel using a complementary radio labeled DNA probe is called …?

Answer 7

Northern blotting

Question 8

Using a radio labeled antibody to identify a protein on a SDS-page gel is called

Answer 8

Western blotting

Question 9

What is needed for PCR

Answer 9

1) Pairs of primers
2) All four DNTPs- A,C,T,G
3) A heat stable DNA polymerase
4) Thermal cycler (machine that cycles between different temperatures.

Question 10

What is the process of PCR cycle

Answer 10

1) Strand separation- 2 strand of the target DNA molecule separated by heat 95c
2) Hybridization of primers- solution is cooled to 54c to allow the primers to hybridize to the 5’ and 3’ ends of the target DNA
3) DNA synthesis - solution is heated to 72c which is optimal temperature for DNA synthesis by Taq DNA polymerase.

Question 11

What is PCR used for?

Answer 11

1) Medical Diagnostics- HIV detect early stage
2) Forensics- DNA amplified Crime identificant
3) Molecular archaeology- evolutionary study

Question 12

Recombinant DNA technology

Answer 12

new combinations of DNA sequences, which can be inserted into various organisms

Question 13

DNA ligase

Requires ?

Answer 13

joins DNA fragments together

• a free 3’- hydroxyl group and a 5’- phosphoryl group
• Both DNA must be double helical
• An energy source such as ATP for joining DNAs

Question 14

Plasmids

Answer 14

circular DNA molecules which can accept novel DNA sequences

Question 15

Phages

Answer 15

viruses that infect bacteria

Question 16

Mutagenesis

Answer 16

making mutations in genes to stud function or to gain new functions

Question 17

Cloning

Answer 17

DNA ligase can be used to insert novel DNA sequences into a DNA vector
- commonly used vectors are plasmids and phages

Question 18

How are vectors prepared for cloning ?

Answer 18

by cutting with a suitable restriction enzyme followed by ligation with target DNA

Question 19

What are the two kinds of vectors used for cloning

Answer 19

Plasmids and Phage

Question 20

What are plasmids?

Answer 20

circular double stranded DNA molecules that occur naturally in some bacteria.

Question 21

What are Phages ?

Answer 21

are viruses that infect bacterial cells and replicate

Question 22

What are the two modes of infection for a Lambda phage

Answer 22

1. Lytic pathway
   - viral functions are fully expressed
   - leads to destruction of the host cell and release of hundred of virus particles
   Lysogenic pathway
   - the phage DNA is integrated into the host genome and can be replicated together with the host DNA

Question 23

What are the pros of Lambda Phage as a Cloning Vector ?

Answer 23

• Phages can tolerate larger DNA insertions than plasmids

- These modified viruses enter bacteria much more easily than plasmid vectors.

Question 24

What are the three kinds of mutations can be made?

Answer 24

1) Deletions
2) Substitutions
3) Insertions

Question 25

A specific sequence within a larger DNA can be excised using ___________ The remaining ends are joined together by _____________ Can use __________ to make targeted deletions of any size ---> overlap extension ______

Answer 25

Restriction enzyme DNA ligase PCR

Question 26

How is mutant protein made by substitution?

Answer 26

it can be made containing a single amino acid substitution using oligonucleotides (primers) with the desired mutation. mutant primer is annealed to the DNA template and is elongated using DNA polymerase.

Question 27

Insertion method?

Answer 27

involves cutting plasmid DNA with two different restriction enzyme to remove a specific region, a new synthesized DNA fragment containing the compatible ends is then ligated into the plasmid and it allows the swapping of one gene for another.

Question 28

What is the process for cloning ?

Answer 28

DNA ligase is used to insert DNA into a DNA vector, the vector is inserted into the host. The vectors are prepared by cutting it with a restriction enzyme followed by ligation with a target DNA (must have compatible ends)

Question 29

How are Plasmids used for cloning?

Answer 29

They carry genes for a selectable marker (antibiotic resistance), Contain a site that tolerates insertion of a new DNA sequence.

Question 30

What are the two selectable markers for pUC18

Answer 30

1. Ampicillin resistance- selects for bacterial cells containing the plasmid 2. Beta- galactosidase- allows for blue/white color selection to determine which bacterial cells contain the DNA insert

Question 31

What are Phages ? What are the two modes of infection of a Lambda phage ?

Answer 31

also called bacteriophages, phages are viral that infect bacterial cells and replicate 1. Lytic pathway: viral functions are fully expressed - leads to destruction of the host cell and release of hundreds of virus particles 2. Lysogenic pathway: the phage DNA is integrated into the host genome and can be replicated together with the host DNA.

Question 32

Why is Lambda phage better used as a vector compared to a plasmid ?

Answer 32

- can tolerate larger DNA insertions than plasmids | - These modified viruses enter bacteria much more easily than plasmid vectors.

Question 33

What is a Genomic library? * look over the creation of Genomic library

Answer 33

It contains a large number of phage particles containing fragments with the entire genome.