MOD 2. Gene Cloning

Question 1

What are the steps in making recombinant DNA?

Answer 1

1. Isolating the DNA
2. Cutting of the DNA
3. Joining the DNA
   * Foreign DNA
   * Vectors
4. Amplifying the recombinant DNA
5. Screening for the Recombinant DNA

Question 2

For Matching Type

Vector

Answer 2

carrier of the gene

Question 2

What are the Requirements of recombinant DNA?

Answer 2

1. gene
2. vector
3. enzymes
4. method to introduce the recombinant DNA to the host cell
5. host cell
6. selection systems

Question 2

For MATCHING TYPE

Gene

what is the use of a gene in recombinant DNA

Answer 2

gene source

Question 3

For MATCHING TYPE

Host cell

Answer 3

allows the replication of the recombinant DNA (bacteria, yeast, insect cells)

Question 4

For MATCHING TYPE

Enzymes

Answer 4

restriction endonucleases, ligase (for “cutting and joining” DNA)

Question 5

For MATCHING TYPE

Selection systems

Answer 5

identify and select the cells containing the recombinant DNA

Question 6

Refers to the set of techniques for recombining genes from different sources in vitro and transferring this recombinant DNA into a cell where it may be expressed.

Answer 6

Recombinant DNA technology

Question 7

• Genetic material of all known organisms
• Nitrogenous base
• Phosphate group
• Pentose- 5- carbon sugar
• The sequence of bases provides the geentic information

Answer 7

Nucleic acids

Question 8

is a unit of inheritance

Answer 8

gene

Question 9

A gene carries the information for a ?

Answer 9

-polypeptide
- structural RNA molecule

Question 10

Made up of structure called nucleotides

A nucleotides consist of 3 things:
* A nitrogenous base, which can be ne

• A five- carbon sugar
• One or more phosphate groups

Answer 10

Deoxyribonucleic- Acid (DNA)

Question 11

Two types of bases

Answer 11

*Purine
*Pyrimidines

* Purines are fused five- and six- membered rings
- Adenine, Guanine
*P

Question 12

Steps in gene cloning (illustrate)

Answer 12

1. a fragment of DNA, conaining the gene to be cloned into a circular DNA molecules called a vector, to produce a recombinant DNA molecule.
2. Transport into the host cell
3. Multiplication of recombinant DNA molecule
4. Division of host cell
5. Numerous cell divisions resulting in a clone

Question 13

Steps for Insulin Production

Example of Genetic Engineering

Answer 13

1. Starter bacterial cell
2. Plasmids extracted
3. Section of DNA cut by enzymes
4. Gene for insulin production cut out with enzymes
5. Gene for insulin inserted into plasmid ting of DNA
6. Modified plasmids put back into the bacterial cells
7. Bacteria multiply producing insulin
8. Insulin extracted from bacteria

Question 14

• Enzymes isolated chiefly from prokaryotes that recognize specific sequences within double stranded DNA.
• Cut DNA within or near to their particular recognition sequences which typically are 4-6 nucleotides in length with a two-fold axis symmetry.

Answer 14

Restriction Endonucleases

Example: EcoRI
Genus: Escherichia
Strain: R

Question 15

For ORIGIN AND FUNCTION

Enzymes that cleave foreign DNA

Answer 15

Bacterial origin

• EcoRI from Esc

*Named after the organism from which they were derived

Question 16

For ORIGIN AND FUNCTION

Function of Bacterium

Answer 16

*Protect bacteria from bacteriophage infection
- Restricts viral replication
* Protect its own DNA by methylating those specific sequence notifs

Question 17

Restrict host range for certin bacteriophage

Answer 17

Restriction enzymes

Question 18

Destroy any “non-self” DNA

Answer 18

Immune system

Question 19

Blank recognizes same sequence in host DNA and protects it by methylating it

Answer 19

Methylase

Question 20

Destroys unprotected = non-self DNA

Answer 20

Restriction enzyme

restriction/modification systems

Question 21

• Specific endonucleases
  *Recognize specific short sequence of DNA and cleave the DNA at or near the recognition sequence.

Answer 21

Restriction Enzymes

Recognition sequences: usually 4 or 6 bases but there are some that are

Question 22

sequence of DNA that is the same when one strand is read from left to right or the other strand is read from right to left– consists of adjacent inverted repeats

Answer 22

Palindrome

Example of Palindrome: GAATTC, CTTAAG

Question 23

# TYPES OF RESTRICTION ENZYME Recognize specific sequences but then track along DNA before cutting one of the strands and releasing a number of nucleotides (-75) where the cut is made. - A second molecule of the endonuclease is required to cut the 2nd strand of the DNA

Answer 23

TYPE 1 | -Therefore random/ imprecise cuts - Not very useful for rDNA applicatio

Question 24

# Types of Resy=triction Enzyme - Recognize a specific target sequence in DNA, and then break the DNA (both strands), within or close to, the recognition site. - - Cuts both strand of DNA within the particular sequence recognized by the restriction enzyme.

Answer 24

TYPE II | DNA sequence = symmetrical ## Footnote Reads the same in the 5'-3' direction on both strands = palindromic -Some enzymes generates "Blunt ends" (cut int he middle)

Question 25

# Types of Restriction Enzymes - Intermediate properties between type Iand type II - Break both DNA strands at a defined distance from a recognition site

Answer 25

TYPE III

Question 26

- Joining linear DNA fragments together with covalent bonds. - Involves creating a phospodiester bond between the 3' OH of one nucleotide and the 5' PO4 of another

Answer 26

Ligation | Catalyze by DNA ligase

Question 27

- Discovered in 1967 by the Gellert, Lehman, Richardson, and Hurwitz laboratories. - Present in all living cells, essential guardians of genomic integrity

Answer 27

DNA ligase

Question 28

Uses of DNA Ligase

Answer 28

*DNA replication *DNA repair 1. nucleotides excision repair 2. base excision repair 3. single-stand breal repair 4. repair of double-strand breaks *Critical reagent in molecular cloning

Question 29

TWO FAMILIES OF DNA LIGASE

Answer 29

1. ATP-dependent ligase 2. NAD- dependent ligases

Question 30

Common in all eukaryal and archeal DNA replication machinery

Answer 30

ATP- dependent ligases | EX. Chlorella virus DNA ligase

Question 31

Found in all bacteria

Answer 31

NAD- dependent ligases | ex. E.coli LigA

Question 32

EXPLAIN THE PROCESS OF MODIFIED BLUNT-END LIGATION

Answer 32

Each method modifies blunt-ended DNA to improve cloning efficiency. Linkers and adaptors create sticky ends, homopolymer tailing promotes complementary base-pairing, and topoisomerase provides a fast and enzyme-driven ligation approach. These methods increase the success rate of cloning and ensure proper insertion of DNA fragments into vectors.

Question 33

a DNA molecule that carries foreign DNA into a host cell, replicates inside the host and produces many copies of itself and the foreign DNA

Answer 33

Cloning Vectors

Question 34

Three Features of a cloning vector

Answer 34

1. Sequence that permit the propagation of itself in the host cell-origin of replication 2. A cloning site to insert foreign DNA 3. A method of selecting for the cells containing the vector with foreign DNA

Question 35

# In Blue white screen Reason of blue colonies

Answer 35

- –active lac-Z gene; with β- galactosidase; in the presence of X- gal, blue color is formed (product)

Question 36

# In Blue white screen Reason of white colonies

Answer 36

inactive lac-Z gene; β galactosidase not synthesized; in the presence of X-gal, no blue color is formed

Question 37

# Antibiotics Commonly Used as Selective Agent Block translocation from amino acyl site to peptidyl site; inactivated by a phosphotransferase

Answer 37

Hygromycin B (HygB)

Question 37

# Antibiotics Commonly Used as Selective Agent Inhibits cell wall formation; inactivated by B-lactamase

Answer 37

Ampicillin (Ap, Amp)

Question 38

# Antibiotics Commonly Used as Selective Agent Binds to 30S subunit and prevents translocation from aminoacyl-tRNA site to peptidyl site; inactivated by a phosphotransferase.

Answer 38

Kanamycin (Km, Kan)

Question 39

# Antibiotics Commonly Used as Selective Agent Bind to 30S subunit and inhibits protein synthesis; inactivated by a phosphotransferase

Answer 39

Neomycin (Nm, Neo)

Question 40

# Antibiotics Commonly Used as Selective Agent Blocks protein intiation complex formation and causes misreading during translation; inactivated by a phosphotransferase

Answer 40

Streptomycin (Sm, Str)

Question 41

# Antibiotics Commonly Used as Selective Agent Prevents binding of aminoacyl- tRNA to 30S ribosomal subunit; resistance gene encodes an inner cell membrane protein that passes the antibiotic out of the cell and blocks the passage of the antibiotic through the cell wall

Answer 41

Tetracycline (Tc, Tet)

Question 42

How can PCR purify a gene?

Answer 42

PCR can purify a gene by selectively amplifying it from a DNA mixture using specific primers. The amplified product is then separated by gel electrophoresis, and the target band is extracted and purified for further use.