10. Gene Manipulation

Question 1

Gene cloning

Answer 1

Isolation and amplification of a given gene

Question 2

Recombinant DNA molecule

Answer 2

DNA molecule made by joining 2+ different DNA molecules

Question 3

Cloning strategy

Answer 3

1. Excise and isolate the gene from a genome
   (Ligation)
2. Clone gene into a vector molecule (transformation)
3. Introduce into host cell that will take it in and reliably replicate it (extraction)
4. Isolate gene from host and retrieve it

Question 4

Restriction endonucleases

Answer 4

Sequence-specific cleavage of DNA by EcoR1 and protection from cleavage by methylation

Make site-specific cuts in DNA

Saves bacteria from phage

Question 5

Types of restriction enzymes

Answer 5

Blunt-end

Overhang

Question 6

Blunt-end cutting enzymes

Answer 6

No leftover nucleotides

Question 7

Overhang cutting enzymes

Answer 7

Provides cohesive ends for re-ligation

Question 8

Restriction sites

Answer 8

Nucleotide sequences

Restriction enzymes commonly recognize palindromic sequences

Provide info on location of genes

Question 9

Restriction analysis

Answer 9

Extract fragment (band)

1. Clone into a vector & propagate
2. Use tag (probe) to molecularly mark region in genome where it is

Question 10

Restriction fragment length polymorphism

Answer 10

Restriction fragments can be used as molecular markers that reveal a blueprint of individual genomes

Question 11

Vectors

Answer 11

Naturally small, transferable, and replicate DNA molecules

Act as template

Question 12

Plasmids

Answer 12

Vector

Can carry over sequences from different genomes

Question 13

Phage DNA

Answer 13

Vector

Used as a vehicle to infect

Question 14

How to clone a gene

Answer 14

1. Isolate DNA fragment w/ gene of interest (in vitro)
2. Insert into plasmid that is a vector (in vitro)
3. Grow in bacteria to amplify (in Vivo)

Question 15

Formation of a recombinant DNA molecule

Answer 15

1. Cut plasmid and DNA with restriction enzyme that will create identical ends (digestion/restriction)
2. Incubate the 2 DNA molecules together in presence of DNA ligase (ligation)
3. Transform to bacteria to propagate and amplify (transformation)

Question 16

Inserting gene into recombinant DNA plasmid

Answer 16

1. Digestion/restriction
2. Ligation
3. Transformation

Can be used to clone many DNA fragments

Question 17

Library

Answer 17

Collection of cloned DNA fragments

Question 18

PCR

Answer 18

Used to amplify DNA in region of interest between 2 primers

Requires knowing the DNA sequences so primers can be designed

Question 19

PCR requires:

Answer 19

1. Template DNA
2. Primers
3. DNTPs
4. Buffer and Mg2
5. DNA polymerase that is het resistant
   I.e., taq polymerase

Question 20

Producing PCR products with sticky ends

Answer 20

DNA sequences introduced to DNA via primers used for PCR

Approach can be used for adding terminal restriction sites that help with cloning PCR product

Question 21

Bacteria=live factories for DNA amplification

Answer 21

Bacteria used to propagate and amplify DNA (e.g., E. Coli)

Question 22

Direction of insert does not matter if

Answer 22

Goal of cloning is to simply amplify its sequence

Question 23

Direction of insertion does matter if

Answer 23

If goal is to express gene under regulation of promoter in vector

Only 1 direction will produce recombinant plasmids that will transcribe sense mRNA

(Reduces efficiency of cloning to 50% of ligation products)

Question 24

Cloning strategies

Answer 24

1. Digestion strategy

2. Ligation strategy

Question 25

Digestion cloning strategy

Answer 25

Choose correct restriction nuclease to use

Question 26

Ligation cloning strategy

Answer 26

Select the type of vector and method of ligation

Question 27

Making selections in cloning

Answer 27

1. Selection for plasmid background

2. Selection for presence of an insert

Question 28

Selection for plasmid background

Answer 28

Ensure that the only surviving bacteria carries a ligation product

Question 29

Selection for presence of an insert

Answer 29

Identify among surviving clones the recombinant plasmids

Question 30

Steps to cloning

Answer 30

1. Insert
2. Digest vector at multi-cloning site
3. Digest insert
4. Ligation insert + plasmid
5. Transform with ligation products
6. Select for either plasmid backbone or presence of an insert

Question 31

Cloning large fragments

Answer 31

Fosmids and BAC’s are cloning vectors that carry large inserts

Question 32

GDNA

Answer 32

DNA libraries that store and study genomes; comprised of individual clones carrying DNA fragments that in association represent part of the whole genome of organism

Question 33

CDNA library

Answer 33

Complementary DNA (DNA generated from RNA by process of reverse transcription

Question 34

How to find clone if interest

Answer 34

1. Use DNA or RNA probes
2. Hybridization of 2 DNA molecules
3. Identify the +’I’ve colony to propagate