Activity No. 6

Question 1

-is the first and crucial technique in the development, performance, and generation of reliable results of all molecular downstream analyses

Answer 1

DNA isolation

Question 2

Downstream analyses examples (6)

PDD RG

Answer 2

polymerase chain reaction (PCR)

DNA sequencing

DNA fingerprinting

restriction fragment length
polymorphism (RFLP)

genomic library construction

Question 3

DNA isolation

• This was first developed by a Swiss physician named____ in _____

Answer 3

Friedrich Miescher

1869

Question 4

FM

he isolated DNA from the cells’ nuclei of leukocytes (from human pus): and named it______ (a name preserved in today’s designation deoxyribonucleic acid).

Answer 4

nuclein

Question 5

the technique was introduced into the clinical laboratory and have produced great changes in diagnostic tools and tests, including detection of pathogens in various samples.

Answer 5

DNA isolation

Question 6

FM

he isolated DNA from the cells’ nuclei of leukocytes (from human pus): and named it______ (a name preserved in today’s designation deoxyribonucleic acid).

Answer 6

nuclein

Question 7

DNA isolation

• This technique involves______ and _______ of DNA from a biological material by utilizing ______ and or ______ methods, in order to obtain highly purified nucleic acids free from any extraction residues and other cellular contents, such as RNA, lipids, polysaccharides and proteins that may adversely molecular downstream analyses.

Answer 7

extraction and purification of DNA

physical and or chemical

Question 8

Examples of extraction residues and other cellular contents, that may adversely molecular downstream analyses.

RLPP

Answer 8

RNA
lipids
polysaccharides
proteins

Question 9

general stages of DNA isolation involve:

Answer 9

1. Cell dissolution
2. Precipitation
3. Purification

Question 9

The isolation of nucleic acids from biological material serves three purposes, namely:

Answer 9

• cell membrane lysis
• inactivation of cellular nucleases
• separation of the desired nucleic acid from cellular debris, proteins, and other substances

Question 10

: the final stage that is done to get a completely pure DNA sample ready to be used.

Answer 10

Purification

Question 11

removal of impurities and proteins from the sample

Answer 11

Precipitation

Question 12

lysis of cell and the nucleus to extract DNA into the buffer

Answer 12

Cell dissolution

Question 13

DNA isolation

the final nucleic acid (DNA) should be free from______, as well as easy to modify for various downstream applications.

Answer 13

impurities

Question 14

it is imperative that isolation should be simple, rapid, and demonstrates increased_____and _____

Answer 14

sensitivity and specificity.

Question 15

it is imperative that isolation should be simple, rapid, and demonstrates increased_____and _____

Answer 15

sensitivity and specificity.

Question 15

DNA isolation

the final nucleic acid (DNA) should be free from______, as well as easy to modify for various downstream applications.

Answer 15

impurities

Question 16

In dealing with clinical specimens (e.g. blood, sputum, urire, other tissues, and body fluids), the following needs to be carefully considered:

Answer 16

• the elimination of potential inhibitors of the DNA polymerase.

• the removal of pathogenicity from hazardous pathogens.

• good target recovery and establishment of the integrity of nucleic acid targets.

Question 17

final target is ______without amplification inhibitors or contaminants such as protein, carbohydrate, and other nucleic acids.

Answer 17

pure nucleic acid

Question 18

DNA extraction method varies, depending on the sample type.

For instance, the DNA extraction method for human blood and other tissues is different from that of_____.

In the same manner,_____ DNA isolation method also has a different protocol from other sample types.

Answer 18

plant

bacterial

Question 19

In particular, bacterial DNA isolation may be carried out using various techniques such as:

Answer 19

1. chemical
   (e.g. Phenol-Chloroform,
   Phenol-Chloroform-Isoamyl, sodium hydroxide, etc.)
2. solid phase (e.g. spin column, etc.)
3. magnetic bead
4. physical (e.g. boiling, etc.) methods.

Question 20

In particular, bacterial DNA isolation may be carried out using various techniques such as:

1. chemical
   (e.g. 3, etc.)
2. solid phase (e.g.1, etc.)
3. magnetic bead
4. physical (e.g.1, etc.) methods.

Answer 20

Phenol-Chloroform,
Phenol-Chloroform-Isoamyl, sodium hydroxide

spin column

boiling

Question 21

• The targets for DNA isolation are diverse.

They can be the cultured bacterial isolates themselves.

Alternatively, one can utilize culture media, including blood culture bottles or various clinical specimens such as sputum stool, urine, tissue, or cerebrospinal fluid.

Question 22

DNA isolation from____ is relatively simple because they are sure colonies and they contain large numbers of organisms.

Answer 22

cultured bacteria

Question 23

_______have ***thick cell wall structures*** due to the several layers of peptidoglycan, making DNA isolation more difficult than it is from gram-____ bacteria possessing thinner walls.

Answer 23

gram-positive bacteria gram-negative

Question 24

(3) are ever more difficult to be broken down due to their ***more complicated structures than Gram-negative and Gram-positive bacteria.***

Answer 24

Cell walls of yeast, mycobacteria, and spores

Question 25

_______have ***thick cell wall structures*** due to the several layers of peptidoglycan, making DNA isolation more difficult than it is from gram-____ bacteria possessing thinner walls.

Answer 25

gram-positive bacteria gram-negative

Question 26

Clinical samples (e.g. blood, urine, sputum, other bod fluids, etc.) may contain all of these types of______. For most of these microbes, the DNA yield and quality can vary considerably depending on the DNA extraction method used.

Answer 26

microorganisms

Question 27

Several strategies have been devised to facilitate lysis of microorganisms, including:

Answer 27

• mechanical • enzymatic • chemical treatments alone or in combination.

Question 28

While some studies cited that mechanical lysis with_____ as well as _______ are probably the ***most universal protocols,*** but these treatments can cause shearing of genomic DNA (gNA) into small fragments that limit the size of potential PCR amplicons and may also cause a bias when different sized amplicons are visualized. This problem becomes exacerbated when samples contain unknown amounts of different types of microbes as each type may require different types of beads and number of leatings.

Answer 28

beads silica column purification

Question 29

_______ and ______ lysis protocols may also be specifically utilized for different types of microbes, however, more time-consuming because several steps are undertaken in these protocols, including enzymatic digestion.

Answer 29

Chemical and enzymatic

Question 30

- Other protocols make use of toxic substances (e.g,_______) that must be safely disposed of or ionic detergents (e.g., SOS) that can react with the sample and must be removed or neutralized to avoid inhibition of subsequent PCR.

Answer 30

guanidine thiocyanate

Question 31

Studies reveal that______ lysis using_____ has been effective for bacterial DNA isolation from different environmental samples such as ***soil, water, air, food, fecal samples, environmental surfaces, as well as broth and plate cultures.***

Answer 31

alkaline; sodium hydroxide

Question 32

Alkaline lysis, which was first Described by____ and_____ in 1979, has been the preferred method for bacterial plasmid DINA isolation.

Answer 32

Bimboim and Doly

Question 33

- This is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells.

Answer 33

Alkaline lysis

Question 34

- It is probably one of the most generally useful techniques because it is a fast, reliable and relatively clean way to obtain DNA from cells.

Answer 34

Alkaline lysis

Question 35

- It is able to rapidly anneal following denaturation. This is what allows the plasmid DNA to be separated from the bacterial chromosome.

Answer 35

Alkaline lysis

Question 36

Alkaline lysis ________ helps to break down the cell wall, as well as disrupts the hydrogen bonding between the DNA bases, thereby converting the double-stranded DNA. (dsDNA) in the cell, including the genomic DNA (gNA) and your plasmid, to ***single-stranded DNA (ssDNA).***

Answer 36

sodium hydroxide

Question 37

This learning activity focuses on bacterial genomic DNA isolation using alkaline lysis method particularly the…

Answer 37

modified polyethylene glycol (PEG)- sodium hydroxide method

Question 38

modified polyethylene glycol (PEG)- sodium hydroxide method developed by (2)

Answer 38

Vingataramin and Frost in 2015.

Question 39

Published protocols employing alkaline lysis typically require additional steps, including______ or other manipulation of the lysates prior to PCR

Answer 39

neutralization

Question 40

- Some published studies reported to have developed an alkaline lysis reagent (2) that would allow a wide variety of biological samples to be used directly in PCR, without the need for further neutralization or DNA isolation steps.

Answer 40

PEG-KOH, PEG-NaOH

Question 41

- The use of PEG at a high concentration (>50% and at alkaline pH meets these criteria. This solution, referred to as the alkaline PEG reagent, originally consists of:

Answer 41

• **60% PEG 200*** (tetra ethylene glycol; Sigma-Aldrich, St. Louis, MO, USA) and • ***20 mM KOH,*** which has a pH of 13.3-13.5.