Name : Soban Sharafat

Roll No : 55

Semester : 3rd

Calss : BS Chemistry
………………………………………….
Submitted to : Mam Fozia

University of Poonch Rawalakot

DNA EXTARATION:

DNA isolation of purification of DNA from sample using a combination of physical

and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher.
[1]
 Currently it is a routine procedure in molecular biology or forensic analyses. For the

chemical method, there are many different kits used for extraction, and selecting the correct

one will save time on kit optimization and extraction procedures. PCR sensitivity detection is

considered to show the variation between the commercial kits.[2]

Basic procedure

There are three basic and two optional steps in a DNA extraction:[3][4]

 Cells which are to be studied need to be collected.

 Breaking the cell membranes open to expose the DNA along with the cytoplasm

within (cell lysis).

o Lipids from the cell membrane and the nucleus are broken down

with detergents and surfactants.

o Breaking down proteins by adding a protease (optional).

o Breaking down RNA by adding an RNase (optional).

 The solution is treated with a concentrated salt solution (saline) to make debris such

as broken proteins, lipids and RNA clump together.

 Centrifugation of the solution, which separates the clumped cellular debris from the

DNA.
 DNA purification from detergents, proteins, salts and reagents used during the cell

lysis step. The most commonly used procedures are:

o Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is

insoluble in these alcohols, it will aggregate together, giving

a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ionic

strength, usually by adding sodium acetate.

o Phenol–chloroform extraction in which phenol denatures proteins in the

sample. After centrifugation of the sample, denatured proteins stay in the organic

phase while the aqueous phase containing nucleic acid is mixed with chloroform to

remove phenol residues from the solution.

o Minicolumn purification that relies on the fact that the nucleic acids may bind

(adsorption) to the solid phase (silica or other) depending on the pH and the salt

concentration of the buffer.

Cellular and histone proteins bound to the DNA can be removed either by adding

a protease or by having precipitated the proteins with sodium or ammonium acetate,

or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

After isolation, the DNA is dissolved in a slightly alkaline buffer, usually in a TE buffer, or

in ultra-pure water.

Method selection[edit]

Some of the most common DNA extraction methods include organic extraction, Chelex

extraction, and solid phase extraction.[5] These methods consistently yield isolated DNA, but
they differ in both the quality and the quantity of DNA yielded. When selecting a DNA

extraction method, there are multiple factors to consider, including cost, time, safety, and

risk of contamination.

Organic extraction involves the addition of and incubation in multiple different chemical

solutions;[5] including a lysis step, a phenol chloroform extraction, an ethanol precipitation,

and washing steps. Organic extraction is often used in laboratories because it is cheap, and it

yields large quantities of pure DNA. Though it is easy, there are many steps involved, and it

takes longer than other methods. It also involves the unfavorable use of the toxic

chemicals phenol and chloroform, and there is an increased risk of contamination due to

transferring the DNA between multiple tubes.[6] Several protocols based on organic extraction

of DNA were effectively developed decades ago,[7] though improved and more practical

versions of these protocols have also been developed and published in the last years.[8]

Chelex extraction method involves adding the Chelex resin to the sample, boiling the

solution, then vortexing and centrifuging it. The cellular materials bind to the Chelex beads,

while the DNA is available in the supernatant.[6] The Chelex method is much faster and

simpler than organic extraction, and it only requires one tube, which decreases the risk of

DNA contamination. Unfortunately, Chelex extraction does not yield as much quantity and

the DNA yielded is single-stranded, which means it can only be used for PCR-based analyses

and not for RFLP.[6]

Solid phase extraction such as using a spin-column based extraction method takes advantage

of the fact that DNA binds to silica. The sample containing DNA is added to a column

containing a silica gel or silica beads and chaotropic salts. The chaotropic salts disrupt the
hydrogen bonding between strands and facilitate binding of the DNA to silica by causing the

nucleic acids to become hydrophobic. This exposes the phosphate residues so they are

available for adsorption.[9] The DNA binds to the silica, while the rest of the solution is

washed out using ethanol to remove chaotropic salts and other unnecessary constituents.
[5]
 The DNA can then be rehydrated with aqueous low salt solutions allowing for elution of

the DNA from the beads.

This method yields high-quality, largely double-stranded DNA which can be used for

both PCR and RFLP analysis. This procedure can be automated[6] and has a high throughput,

although lower than the phenol-chloroform method. This is a one-step method i.e the entire

procedure is completed in one tube. This lowers the risk of contamination making it very

useful for forensic extraction of DNA. Multiple solid phase extraction commercial kits are

manufactured and marketed by different companies; the only problem is that they are more

expensive than organic extraction or Chelex extraction.

Special types[edit]

Specific techniques must be chosen for isolation of DNA from some samples. Typical

samples with complicated DNA isolation are:

 archaeological samples containing partially degraded DNA, see ancient DNA [10]

 samples containing inhibitors of subsequent analysis procedures, most notably

inhibitors of PCR, such as humic acid from soil, indigo and other fabric dyes

or haemoglobin in blood

 samples from microorganisms with thick cellular wall, for example yeast

 samples containing mixed DNA from multiple sources

Extrachromosomal DNA is generally easy to isolate, especially plasmids may be easily

isolated by cell lysis followed by precipitation of proteins, which traps chromosomal DNA in

insoluble fraction and after centrifugation, plasmid DNA can be purified from soluble

fraction.

A Hirt DNA Extraction is an isolation of all extrachromosomal DNA in a mammalian cell.

The Hirt extraction process gets rid of the high molecular weight nuclear DNA, leaving only

low molecular weight mitochondrial DNA and any viral episomes present in the cell.

Detection of DNA[edit]

Main article: Quantification of nucleic acids

A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure

involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction

requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the

2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the

compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be

determined measuring the intensity of absorbance of the solution at the 600 nm with

a spectrophotometer and comparing to a standard curve of known DNA concentrations.

Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280

nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres,

and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has a ratio of 1.8 at
260/280 and is relatively free from protein contamination. A DNA preparation that is

contaminated with protein will have a 260/280 ratio lower than 1.8.

DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an

agarose gel, staining with ethidium bromide (EtBr) or a different stain and comparing the

intensity of the DNA with a DNA marker of known concentration.

Using the Southern blot technique, this quantified DNA can be isolated and examined further

using PCR and RFLP analysis. These procedures allow differentiation of the repeated

sequences within the genome. It is these techniques which forensic scientists use for

comparison, identification, and analysis.

Backcrossing

Backcrossing is a crossing of a hybrid with one of its parents or an individual genetically

similar to its parent, in order to achieve offspring with a genetic identity which is closer to that of

the parent. It is used in horticulture, animal breeding and in production of gene

knockout organisms.

Backcrossed hybrids are sometimes described with acronym "BC", for example, an F1

hybrid crossed with one of its parents (or a genetically similar individual) can be termed a BC1

hybrid, and a further cross of the BC1 hybrid to the same parent (or a genetically similar

individual) produces a BC2 hybrid.[1]

Dvantages

 If the recurrent parent is an elite genotype, at the end of the backcrossing programme

an elite genotype is recovered.

 As there is no "new" recombination, the elite combination is not lost.

Disadvantages

 Works poorly for quantitative traits

 Is more restricted for recessive traits

 In practice, sections of genome from the non-recurrent parents are often still present

and can have unwanted traits associated with them

 For very wide crosses, limited recombination may maintain thousands of ‘alien’ genes

within the elite cultivar

 Many backcrosses are required to produce a new cultivar which can take many years

Natural backcrossings

York radiate groundsel (Senecio eboracensis) is a naturally occurring hybrid species of

Oxford ragwort (Senecio squalidus) and common groundsel (Senecio vulgaris). It is thought

to have arisen from a backcrossing of the F1 hybrid with S. vulgaris.[2]

Again, the pure tall (TT) and pure dwarf (tt) pea plants when crossed in the parental

generation, they produce all heterozygote (Tt) tall pea plants in the first filial generation. The

cross between first filial heterozygote tall (Tt) pea plant and pure tall (TT) or pure dwarf (tt)

pea plant of the parental generation is also an example for the back-crossing between two

plants. In this case, the filial generation formed after the back cross may have a phenotype

ratio of 1:1 if the cross is made with recessive parent or else all offspring may be having

phenotype of dominant trait if back cross is with parent having dominant trait. The former of

these traits is also called a test cross.

Drosophila
Drosophila (/drəˈsɒfɪlə, drɒ-, droʊ-/[1][2]) is a genus of flies, belonging to

the family Drosophilidae, whose members are often called "small fruit flies" or (less

frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many

species to linger around overripe or rotting fruit. They should not be confused with

the Tephritidae, a related family, which are also called fruit flies (sometimes referred to as "true

fruit flies"); tephritids feed primarily on unripe or ripe fruit, with many species being regarded as

destructive agricultural pests, especially the Mediterranean fruit fly.

One species of Drosophila in particular, D. melanogaster, has been heavily used in

research in genetics and is a common model organism in developmental biology. The terms

"fruit fly" and "Drosophila" are often used synonymously with D. melanogaster in modern

biological literature. The entire genus, however, contains more than 1,500 species [3] and is very

diverse in appearance, behavior, and breeding habitat.

Etymology

The term "Drosophila", meaning "dew-loving", is a modern

scientific Latin adaptation from Greek words δρόσος, drósos, "dew",

and φίλος, phílos, "loving" with the Latin feminine suffix -a.

Morphology

Drosophila species are small flies, typically pale yellow to reddish brown to black,

with red eyes. When film of lenses (eyes) are removed, the drosophila brain is
revealed. Drosophila brain structure and function develop and age significantly

from larval to adult stage. Developing brain structures make these flies a prime

candidate for neuro-genetic research.[4] Many species, including the noted

Hawaiian picture-wings, have distinct black patterns on the wings. The plumose

(feathery) arista, bristling of the head and thorax, and wing venation are characters

used to diagnose the family. Most are small, about 2–4 mm long, but some,

especially many of the Hawaiian species, are larger than a house fly.

Habitat

Drosophila species are found all around the world, with more species in the

tropical regions. Drosophila made their way to the Hawaiian Islands

and radiated into over 800 species.[5] They can be found in deserts, tropical

rainforest, cities, swamps, and alpine zones. Some northern species hibernate. The

northern species D. montana is the best cold-adapted,[6] and is primarily found at

high altitudes.[7] Most species breed in various kinds of decaying plant

and fungal material, including fruit, bark, slime fluxes, flowers, and mushrooms.

The larvae of at least one species, D. suzukii, can also feed in fresh fruit and can

sometimes be a pest.[8] A few species have switched to being parasites or predators.

Many species can be attracted to baits of fermented bananas or mushrooms, but

others are not attracted to any kind of baits. Males may congregate at patches of
suitable breeding substrate to compete for the females, or form leks, conducting

courtship in an area separate from breeding sites.[citation needed]

Several Drosophila species, including D. melanogaster, D. immigrans, and D.

simulans, are closely associated with humans, and are often referred to

as domestic species. These and other species (D. subobscura, Zaprionus indianus[9]

[10][11]
) have been accidentally introduced around the world by human activities such

as fruit transports.

Reproduction

Males of this genus are known to have the longest sperm cells of any studied

organism on Earth, including one species, Drosophila bifurca, that has sperm cells

that are 58 mm (2.3 in) long.[12] The cells are mostly tail, and are delivered to the

females in tangled coils. The other members of the genus Drosophila also make

relatively few giant sperm cells, with that of D. bifurca being the longest.[13] D.

melanogaster sperm cells are a more modest 1.8 mm long, although this is still

about 35 times longer than a human sperm. Several species in the D.

melanogaster species group are known to mate by traumatic insemination.

Salivary gland
The salivary glands in mammals are exocrine glands that produce saliva through a

system of ducts. Humans have three paired major salivary glands (parotid, submandibular,

and sublingual) as well as hundreds of minor salivary glands.[1] Salivary glands can be classified

as serous, mucous or seromucous (mixed).

In serous secretions, the main type of protein secreted is alpha-amylase, an enzyme that breaks

down starch into maltose and glucose,[2] whereas in mucous secretions the main protein secreted

is mucin, which acts as a lubricant.[1]

In humans, between 0.5 and 1.5 litres of saliva are produced every day. [3] The secretion of saliva

(salivation) is mediated by parasympathetic stimulation; acetylcholine is the

active neurotransmitter and binds to muscarinic receptors in the glands, leading to increased

salivation.

Structure
Parotid glands

The two parotid glands are major salivary glands wrapped around

the mandibular ramus in humans.[5] These are largest of the salivary glands,

secreting saliva to facilitate mastication and swallowing, and amylase to begin the

digestion of starches.[6] It is the serous type of gland which secretes alpha-

amylase (also known as ptyalin).[7] It enters the oral cavity via the parotid duct. The

glands are located posterior to the mandibular ramus and anterior to the mastoid
process of the temporal bone. They are clinically relevant in dissections of facial

nerve branches while exposing the different lobes, since any iatrogenic lesion will

result in either loss of action or strength of muscles involved in facial expression.

[7]
 They produce 20% of the total salivary content in the oral cavity.[6] Mumps is

a viral infection, caused by infection in the parotid gland.[8]

Submandibular glands

The submandibular glands (previously known as submaxillary glands) are a

pair of major salivary glands located beneath the lower jaws, superior to

the digastric muscles.[5] The secretion produced is a mixture of both serous

fluid and mucus, and enters the oral cavity via the submandibular duct or Wharton

duct.[6] Approximately 65-70% of saliva in the oral cavity is produced by the

submandibular glands, even though they are much smaller than the parotid glands.
[6]
 This gland can usually be felt via palpation of the neck, as it is in the superficial

cervical region and feels like a rounded ball. It is located about two fingers above

the Adam's apple (laryngeal prominence) and about two inches apart under the

chin.

Sublingual glands

The sublingual glands are a pair of major salivary glands located inferior to

the tongue, anterior to the submandibular glands.[5] The secretion produced is

mainly mucous in nature; however, it is categorized as a mixed gland.[7] Unlike the

other two major glands, the ductal system of the sublingual glands does not have

intercalated ducts and usually does not have striated ducts either, so saliva exits

directly from 8-20 excretory ducts known as the Rivinus ducts.[7] Approximately

5% of saliva entering the oral cavity comes from these glands.[6]

Minor salivary glands

There are 800 to 1,000 minor salivary glands located throughout the oral

cavity within the submucosa[9] of the oral mucosa in the tissue of the buccal, labial,

and lingual mucosa, the soft palate, the lateral parts of the hard palate, and the floor

of the mouth or between muscle fibers of the tongue.[10] They are 1 to 2 mm in

diameter and unlike the major glands, they are not encapsulated by connective

tissue, only surrounded by it. The gland has usually a number of acini connected in

a tiny lobule. A minor salivary gland may have a common excretory duct with

another gland, or may have its own excretory duct. Their secretion is

mainly mucous in nature and have many functions such as coating the oral cavity

with saliva. Problems with dentures are sometimes associated with minor salivary

glands if there is dry mouth present (see further discussion). [9] The minor salivary

glands are innervated by the seventh cranial or facial nerve.[10]

Von Ebner's glands

Von Ebner's glands are glands found in a trough circling the circumvallate

papillae on the dorsal surface of the tongue near the terminal sulcus. They secrete a

purely serous fluid that begins lipid hydrolysis. They also facilitate the perception

of taste through secretion of digestive enzymes and proteins.[9] The arrangement of

these glands around the circumvallate papillae provides a continuous flow of fluid

over the great number of taste buds lining the sides of the papillae, and is important

for dissolving the food particles to be tasted.

Nerve supply

Salivary glands are innervated, either directly or indirectly, by

the parasympathetic and sympathetic arms of the autonomic nervous system.

Parasympathetic stimulation evokes a copious flow of saliva.

 Parasympathetic innervation to the salivary glands is carried via cranial

nerves. The parotid gland receives its parasympathetic input from

the glossopharyngeal nerve (CN IX) via the otic ganglion,[11] while the

submandibular and sublingual glands receive their parasympathetic input from

the facial nerve (CN VII) via the submandibular ganglion.[12] These nerves

release acetylcholine and substance P, which activate the IP3 and DAG

pathways respectively.
 Direct sympathetic innervation of the salivary glands takes place via

preganglionic nerves in the thoracic segments T1-T3 which synapse in

the superior cervical ganglion with postganglionic neurons that release

norepinephrine, which is then received by β1-adrenergic receptors on the acinar

and ductal cells of the salivary glands, leading to an increase in cyclic

adenosine monophosphate (cAMP) levels and the corresponding increase of

saliva secretion. Note that in this regard both parasympathetic and sympathetic

stimuli result in an increase in salivary gland secretions[13], the difference lies on

the composition of this saliva, once sympathetic stimulus results particularly in

the increase of amilase secretion, which is produced by serous glands. The

sympathetic nervous system also affects salivary gland secretions indirectly by

innervating the blood vessels that supply the glands, resulting in

vasoconstriction through the activation of α1 adrenergic receptors, lessening the

saliva's water content.

Microanatomy

The gland is internally divided into lobules. Blood vessels and nerves enter the

glands at the hilum and gradually branch out into the lobules.

Acini

Secretory cells are found in a group, or acinus (plural, acini). Each acinus is

located at the terminal part of the gland connected to the ductal system, with many

acini within each lobule of the gland. Each acinus consists of a single layer of

cuboidal epithelial cells surrounding a lumen, a central opening where the saliva is

deposited after being produced by the secretory cells. The three forms of acini are

classified in terms of the type of epithelial cell present and the secretory product

being produced: serous, mucoserous and mucous.[14][15]

Ducts

In the duct system, the lumina are formed by intercalated ducts, which in

turn join to form striated ducts. These drain into ducts situated between the lobes of

the gland (called interlobar ducts or secretory ducts). These are found on most

major and minor glands (exception may be the sublingual gland).[14]

All of the human salivary glands terminate in the mouth, where the saliva

proceeds to aid in digestion. The saliva that salivary glands release is quickly

inactivated in the stomach by the acid that is present, however saliva also contains

enzymes that are actually activated by stomach acid.

Gene and protein expression

About 20,000 protein coding genes are expressed in human cells and 60% of

these genes are expressed in normal, adult salivary glands.[16][17] Less than 100

genes are more specifically expressed in the salivary gland. The salivary gland

specific genes are mainly genes that encode for secreted proteins and compared to

other organs in the human body; the salivary gland has the highest fraction of

secreted genes. The heterogeneous family of proline-rich, human salivary

glycoproteins, such as PRB1 and PRH1, are salivary gland specific proteins with

highest level of expression. Examples of other specifically expressed proteins

include the digestive amylase enzyme AMY1A, the mucin MUC7 and statherin, all

of major importance for specific characteristics of saliva.