Biotechnology (W16) - Recombinant DNA technology

Question 1

The ability of researcher’s to manipulate DNA at the molecular level is known as _________ (more than one word).

Answer 1

Recombinant DNA technology or genetic engineering

Question 2

The term _________ ___ refers to the ability to prepare novel DNA species by recombining DNA from different sources.

Answer 2

recombinant DNA

Question 3

Most of the tools of recombinant DNA technology have arisen from __________ and _______.

Answer 3

bacteriophage and bacteria

Question 4

______ are small, circular double-stranded DNA molecules that are common in bacteria and which have been adapted to become key ______ for recombinant DNA.

Answer 4

1 - Plasmids

2 - vector

Question 5

_______ ________ are enzymes that cleave dsDNA at specific sites called _________ _________.

Answer 5

1 - Restriction endonucleases

2 - recognition sites

Question 6

Bacteria use _________ __________ as a defense mechanism.

Answer 6

restriction endonucleases

Question 7

In recombinant DNA technology, _________ __________ are used as molecular scissors to cleave ____ molecules to allow them to be recombined with other ____ molecules.

Answer 7

1 - restriction endonucleases
2 - DNA
3 - DNA

Question 8

DNA and RNA polymerases synthesize nucleic acids in a __________-dependent manner.

Answer 8

template

Question 9

DNA ligases catalyze the formation of ____________ bonds to allow assembly of _________ ____ constructs.

Answer 9

1 - phosphodiester bonds

2 - recombinant DNA

Question 10

What is cDNA?

Answer 10

Double stranded DNA copies of RNA

Question 11

Expression of proteins using recombinant technology affords the ability to: (2 things)

Answer 11

1 - ovexpress proteins for the purpose of purifying them
2 - selectively mutate one or more residues in the protein for the purposes of structure/function analyses of the protein

Question 12

A technique known as _____-__________ __________ is performed to introduce the desired mutations into the cloned cDNA and is used to alter nucleotide sequence of cloned DNA at a predefined position or site.

Answer 12

site-directed mutagenesis.

Question 13

SDM can be used to change only a single or multiple _____-_____, or can be used to create more extensive sequences changes such as ______ and ________ or ______ or ________ of restriction enzyme sites.

Answer 13

1 - base-pairs
2 - insertions and deletions
3 - removal or introduction

Question 14

There are two general methods for oligonucleotide site-directed mutagenesis. What are they?

Answer 14

1 - Synthetic DNA casettes

2 - Enzymatic extensions of a mutagenic oligonucleotide (also called SDM)

Question 15

What is the end result of both methods of oligonucleotide SDM?

Answer 15

an oligonucleotide encoding the new DNA sequence is inserted into a cloned DNA fragment

Question 16

What method will we be using?

Answer 16

Enzymatic extension of a mutagenic oligonucleotide

Question 17

The DNA polymerases used for the method we are using are?

Answer 17

Bacteriophage T4 or R7.

Question 18

Why are we using bacteriophage T4 or R7?

Answer 18

Do not proofread mutagenic oligonucleotides therefore making it possible to obtain mutant plasmids

Question 19

mutant DNA is also called?

Answer 19

heteroduplex DNA

Question 20

How is the system able to remove the template DNA prior to bacterial transformation?

Answer 20

pcr product is digested with the endonuclease Dpn1

Question 21

What does the endonuclease Dpn1 do?

Answer 21

digests methylated sequences in the template DNA

Question 22

The sequence of interest (the newly created mutant DNA) is transformed into competent cells which are able to perform _____ ______.

Answer 22

nick repair

Question 23

Draw the SDM process.

Answer 23

Refer to notes.
1 - template DNA (plasmid vector) is denatured to become ss
2 - anneal mutagenic oligonucleotide to ss template DNA
3 - Add DNA pol (T4/R7), DNA ligase, ATP and dNTPs
4 - Synthesis of complementary strand
5 - DNA ligase joins end, have heteroduplex DNA (mixture of both wt and mutant DNA)
6 - Remove parental DNA by Dpn1 methylated DNA digestion
7 - Transform into competent cells that perform nick-repair

Question 24

The wt sequences need to be long enough so a stable DNA duplex can form between the ___________ _____ and the template DNA.

Answer 24

oligonucleotide primer