Key Recombinant DNA Techniques Impacting On Medicine

Question 1

Definition of Recombinant DNA ?

Answer 1

DNA molecule made in the laboratory using at least two different sources of DNA.

Question 2

What is the first recombinant DNA ?

Answer 2

1973

plasmids from E. coli.

Question 3

Function of Restriction Enzymes (RE) ?

Answer 3

cleave double-stranded DNA into manageable fragments.

Question 4

What is the Most powerful tools in molecular biology?

Answer 4

Restriction Enzymes (RE)

Question 5

Source of Restriction enzymes?

Answer 5

Are made by microbes as a defense mechanism against
viruses or other “foreign” DNA.

Bind to DNA at specific sequences and cut it (act as
“molecular scissors”).

Question 6

The place where Restriction enzymes cut within specific

palindromic sequences called?

Answer 6

restriction

sites,

Question 7

What type of bond the Restriction enzymes cut ?

Answer 7

phosphodiester

Question 8

Example of restriction enzyme ?

And where it cuts

Answer 8

EcoR1
Find the sequence GAATTC
And cut between GA
Sticky ends (Staggered cuts)

Question 9

Type of cleaving by restriction enzymes ?

Answer 9

staggered cuts sticky” ends

blunt” ends.

Question 10

DNA molecules from different species are cut
By same restriction enzyme.
Or diff restriction enzyme.
?

Answer 10

same restriction enzyme.

Question 11

Two sticky end can bind together by which bond ?

Answer 11

Hydrogen boun

Question 12

The staining for DNA fragments ?

Answer 12

Ethidium

Bromide

Question 13

DNA fragments can be separated by ?

Answer 13

electrophoresis

Question 14

Transformation meaning ?

Answer 14

Recombinant DNA is cloned by

inserting it into host cells

Question 15

if host cells

are from an we called it ?

Answer 15

Transfection

Question 16

transgenic meaning

Answer 16

altered host cell is called

Question 17

Example of host cells ?

Answer 17

Bacteria, yeasts, and cultured plant cells

Question 18

Characteristics of Vectors it must contain

Answer 18

A replication unit (for propagation in host cells),
✓ Recognition sequences for restriction enzymes
(at which to insert new DNA), and
✓ Genetic (reporter) markers to identify their
presence in host cells.

Question 19

Example of vector ?

Answer 19

Plasmid pBR322

Question 20

Vector example ?

Answer 20

✓ Virus (act as a “Trojan horse”)
✓ Chemical treatment, such as high Ca2+
✓ Liposomes (lipid coating over DNA)
✓ Electroporation (high-voltage current pulses )
✓ Microinjection
✓ Bombardment with DNA coated particles

Question 21

Method of identify which cells contain the vector.

Answer 21

nutritional, antibiotic resistance, or fluorescent markers

Question 22

How to mark a Recombinant DNA ?

Answer 22

Inactivating a Gene

Question 23

What is the (genomic) library?

Answer 23

represents all genes of an

organism

Question 24

Source of gene library Constructing Libraries

.?

Answer 24

genomic DNA

complementary
DNA (cDNA).

Synthesis gene

Question 25

What we can use to create | and amplify a specific cDNA sequence.?

Answer 25

Reverse transcriptase and PCR

Question 26

What is the PCR?

Answer 26

is in vitro technique for | generating large quantities of a specified DNA.

Question 27

What is the molecular photocopier.

Answer 27

PCR

Question 28

Steps of PCR?

Answer 28

1. Denaturation 2. Annealing 3. Synthesis (chain extension)

Question 29

Materials Required for PCR?

Answer 29

1- target DNA fragment. 2- Two synthetic primers (forward and reverse), GC content between 40% to 60%. 3- thermostable DNA polymerase (Taq polymerase) which can sustain high temperature. 4- A large number of free deoxynucleotides (dNTPs) 5- Divalent Cations (Mg2+): Required for optimal polymerase activity. 6- Buffer to maintain pH.

Question 30

Applications of PCR

Answer 30

PCR is highly sensitive Analysis of Mutation. • Amplification of cloned DNA. • Production of DNA for sequencing. • PCR in Forensic science. • Detection of bacteria and viruses. • Study of evolution from DNA of archeological samples

Question 31

Meaning of DNA Sequencing?

Answer 31

finding the order of | nucleotides on a single strand of DNA .

Question 32

The Sanger’s Technique | Depends on what ?

Answer 32

dideoxynucleotides i.e ddNTPs (dideoxyadenine, | dideoxyguanine, etc).

Question 33

Requirements of Sanger’s Technique | ?

Answer 33

1- Multiple copies of template DNA strand. 2-radioactive tagged primer (a small piece of DNA that can pair with the template DNA to act as a starting point for replication). 3-DNA polymerase 4-four deoxynucleotides (dNTPs) 5-proportion of each of the four labeled dideoxynucleotides (ddNTPs)

Question 34

The sequence is read in Sanger’s Technique | from? I

Answer 34

bottom up,

Question 35

An advancement of the sanger’s method

Answer 35

is the use of automated sequencing machines using ddNTPs that are each tagged with a different color fluorescent dye. • Instead of performing four different reactions, DNA synthesis occurs in one tube.