L5

Question 1

What are the Two R-DNA techniques that are commonly used

Answer 1

Polymerase chain reaction (PCR)
DNA sequencing

Question 2

Applications of R-DNA techniques

Answer 2

1) investigate molecular pathogenesis
2) develop diagnostic tests
3) develop treatment strategies

Question 3

PCR and DNA sequencing are based on 2 principles what are they?

Answer 3

DNA synthesis
Nucleic acid hybridization

Question 4

Amplifies a target DNA in large amount

Answer 4

PCR

Question 5

DNA synthesis done repeatedly for multiple times

Answer 5

PCR

Question 6

PCR pros (3)

Answer 6

1- super sensitive and fast
Target any region using a pair of primers (flexible)
DNA is increased

Question 7

PCR is based on 2 principles

Answer 7

DNA synthesis
Nucleic acid hybridization

Question 8

Requirements of PCR

Answer 8

1- template
2- primer
3- dNTP
4- enzymes

Question 9

What are the characteristics of PCR primers?

Answer 9

-Single stranded DNA molecules

-Complementary to both strands of the template

-define the region to be amplified

Question 10

What enzyme is used in PCR?

Answer 10

Taq DNA polymerase

Question 11

Why will The template (λDNA) will not be visible on the gel

Answer 11

because of its minute amount

Question 12

How many cycles does PCR require

Answer 12

20-40

Question 13

What is the role of denaturation

Answer 13

Separate DNA strand at 94°C

Question 14

What is the role of annealing

Answer 14

Ensure primer binding, Temperature is selected based on the primer size

Question 15

Extension is done at how many degrees Celsius

Answer 15

74°C

Question 16

Why do we target a selection by the primer

Answer 16

So only the selected region is amplified

Question 17

What is the fate of the primer in PCR

Answer 17

Becomes part of the newly synthesized stand

Question 18

At is important for

Answer 18

Primer binding

Question 19

What is the At

Answer 19

Tm of primer -5C

Question 20

Tm of the primer depends on

Answer 20

Size + base composition

Question 21

Tm formula

Answer 21

Tm = 2(A+T) + 4(C+G)C

Question 22

What are the applications of PCR

Answer 22

Disease mechanism
Disease diagnosis
Treatment development
DNA sequencing

Question 23

What is PCR limitation

Answer 23

Sequence of the target DNA molecule must be known

Question 24

What are the three methods for DNA sequencing and what are their needs

Answer 24

1- maxam gilbert : chemical

2- sanger’s method: enzymatic
* template
*dNTP
*ddNTP
*primer
*electrophoresis

3-pyrosequencing: enzymatic
*dNTP
*primer

Question 25

What is the Principle of Sanger’s DNA sequencing

Answer 25

Base selective termination of dna synthesis

Question 26

In a single reaction of DNA sequencing, both strands cannot by sequenced simultaneously why?

Answer 26

Because the primer is complementary to 1 strand only

Question 27

DNA sequencing is accomplished in two steps

Answer 27

1- generating a sequence ( synthesizing new strands)

2- obtain sequence ( separation of fragments by capillary electrophoresis)

Question 28

In DNA S, the color indicates the ___ while the size indicates the __

Answer 28

Base
Position

Question 29

In capillary electrophoresis, separation is based on the ___, while the autodetector is according to the __

Answer 29

Size
Color

Question 30

How many cycles are needed for capilary reactions

Answer 30

40, beacuse the product doesn’t act as a template

Question 31

What are the uses of dna sequencing

Answer 31

1. Identification of mutations→disease diagnosis and disease mechanism
2. Study of gene structure and functions
3. Gene cloning