MOLECULAR BIOLOGY

MOLECULAR BIOLOGY

Molecular biology; the study of biology at the molecular level.

Molecular biology; the study of gene structure and functions at the molecular level to understand the molecular basis of hereditary, genetic variation, and the expression patterns of genes.

The structure of DNA was described by British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti-parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases.

This structure explains how genes engage in replication, carrying information and acquiring mutation.

The G+C content of a natural DNA can vary from 22-73% and this can have a strong effect on the physical properties of DNA, particularly its melting temperature.

DNA and RNA are long chain polymers of small compound called nucleotides.

Each nucleotide is composed of

a base; sugar (ribose in RNA or

deoxyribose in DNA) and a phosphate group.

General Structure of Nucleic Acid

There are four different types of nucleotides found in DNA, differing only in the nitrogenous base: A is for adenine; G is for guanine; C is for cytosine and T is for thymine.

These bases are classified based on their chemical structures into two groups: adenine and guanine are double ringed structure termed purine , thymine and cytosine are single ring structures termed pyrimidine.

The bases pair in a specific way:

Adenine A with Thymine T (two hydrogen bonds) and

Guanine G with Cytosine C (three hydrogen bonds).

Strands in the DNA runs antiparallel

FORM OF DNA

Characteristic A-DNA B-DNA Z-DNA

diameter (D) 2.6 nm 2.0 nm 1.8 nm

bp/turn 11 10.4 12

degrees rotation/bp +32.7 +34.6 -30.0

axial distance/turn 2.8 nm 3.4 nm 4.5 nm

axial distance between bp 0.25 nm 0.33 nm 0.38 nm

Replication proceeds in a semiconservative manner, each strand of the DNA

helix serves as a template for the synthesis of complementary DNA strands. This lead to the formation of two complete copies of the DNA molecule, each consisting of one strand derived from the parent DNA molecule and one newly synthesized complementary strand.

GENOMIC DNA ORGANIZATION

Eukaryotic genes: DNA molecules complexed with other proteins especially basic proteins called histones, to form a substance known as chromatin.

EUKARYOTIC CHROMATIN

Eukaryotic chromatin is folded in several ways. The first order of folding involves structures called nucleosomes, which have a core of histones, around which the DNA winds ( four pairs of histones H2A, H2B,H3 and H4 in a wedge shaped disc, around it wrapped a stretch of 147 bp of DNA).

FUNCTION OF THE DNA

Deoxyribonucleic Acid (DNA), the gigantic molecule which is used to encode genetic information for all life on Earth excepts some viruses.

The chemical basis of hereditary and genetic variation are related to DNA.

DNA directs the synthesis of RNA which in turn directs protein synthesis.

Central Dogma of Molecular Biology

The flow of genetic information as follows:

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DNA ISOLATIONOBJECTIVES

To understand the basic process of isolation of DNA from various sources eg blood, tissue, bacteria.

To realise that different types of DNA require different methods of isolation.

To realise that the method used is dependent upon the final application.

To understand the basis of gel electrophoresis To realise that there are different types of gel

electrophoresis.

DNA EXTRACTION METHODS

Introduction Deoxyribonucleic acid (DNA) isolation is an extraction process of DNA from various sources. Methods used to isolate DNA are dependent on the source, age, and size of the sample. Despite the wide variety of methods used, there are some similarities among them. In general, they aim to separate DNA present in the nucleus of the cell from other cellular components.

Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or forensic purposes. Scientists use DNA in a number of applications, such as introduction of DNA into cells and animals or plants, or for diagnostic purposes. In medicine the latter application is the most common. On the other hand, forensic science needs to recover DNA for identification of individuals (for example rapists, petty thieves, accident, or war victims), paternity determination, and plant or animal identification

Sources for DNA isolation are very diverse. Basically it can be isolated from any living or dead organism. Common sources for DNA isolation include whole blood, hair, sperm, bones, nails, tissues, blood stains, saliva, buccal (cheek) swabs, epithelial cells, urine, paper cards used for sample collection, bacteria, animal tissues, or plants.

How Can We Recover DNA From a Variety of Sources

of Biological Evidence?BloodSemenSalivaUrineHair (w/Root & Shaft)TeethBoneTissue

Cigarette ButtsEnvelope & StampsFingernail ClippingsChewing GumBite MarksFeces

WHAT ARE THE ESSENTIAL COMPONENTS OF A DNA EXTRACTION PROCEDURE?

1. Maximize DNA recovery2. Remove inhibitors3. Remove or inhibit

nucleases4. Maximize the quality of

DNA

WHAT ARE THE MOST COMMONLY USED DNA EXTRACTION PROCEDURES IN FORENSIC SCIENCE?

Organic (Phenol-Chloroform) Extraction Non-Organic (Proteinase K and Salting

out) Chelex (Ion Exchange Resin) Extraction FTA Paper (Collection, Storage, and

Isolation)The method utilized may be sample dependant, technique dependant, or analyst preference

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SPECIFIC METHODS OF DNA ISOLATION

Genomic DNA SDS/Proteinase K Qiagen columns Alkaline method Automated methods

Plasmid DNA Alkaline/SDS Qiagen column methods

Bacteriophage M13 DNA PEG precipitaton method

Bacteriophage lambda DNA PEG/Salt precipitation method

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ISOLATION OF DNAMETHODS OF ISOLATING DNA

Cell extraction Organic - phenol, CHCl3 high salt guanidinium HCl

Removal of cell debris proteins, lipids, polysaccharides

Concentration of DNA ethanol, isopropranol DNA absorbing matrix CTAB, spermidine

Optional steps Rnase A removal of RNA

ORGANIC EXTRACTION

Perhaps the most basic of all procedures in forensic molecular biology is the purification of DNA. The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform.

Presence of proteins, lipids, polysaccharides and some other organic or inorganic compounds in the DNA preparation can interfere with DNA analysis methods, especially with polymerase chain reaction (PCR). They can also reduce the quality of DNA leading to its shorter storage life

ORGANIC EXTRACTION PROCEDURE

Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet nuclei.

Resuspend nuclei, add Sodium Dodecly Sulfate (SDS), Proteinase K. Lyse nuclear membrane and digest protein.

DNA released into solution is extracted with phenol-chloroform to remove proteinaceous material.

DNA is precipitated from the aqueous layer by the additional of ice cold 95% ethanol and salt

Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.

REAGENTS Cell Lysis Buffer - Non-ionic

detergent, Salt, Buffer, EDTA designed to lyse outer cell membrane of blood and epithelial cells, but will not break down nuclear membrane.

EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.

REAGENTS Proteinase K - it is usual to remove

most of the protein by digesting with proteolytic enzymes such as Pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.

PURPOSE OF DNA EXTRACTION

To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc

Break down the cell wall and membranes

Centrifuge to separate the solids from the dissolved DNA

Precipitate the DNA using isopropanol

Centrifuge to separate the DNA from the dissolved salts and sugarsWash the

DNA pellet with Ethanol and dry the pellet

Dissolve DNA

Overview of DNA Extraction

BASIC PROTOCOL

Most DNA extraction protocols consist of two parts1. A technique to lyse the cells gently and

solubilize the DNA2. Enzymatic or chemical methods to remove

contaminating proteins, RNA, or macromolecules

In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell.

1. Lysis2. Precipitation3. Wash4. Resuspension

LYSIS:IN DNA EXTRACTION FROM PLANTS, THIS STEP COMMONLY REFERS TO THE BREAKING OF THE CELL WALL AND CELLULAR MEMBRANES (MOST IMPORTANTLY, THE PLASMA AND NUCLEAR MEMBRANES)

The cell wall (made of cellulose) is disrupted by mechanical force (for example, grinding the leaves)

Then the addition of a detergent in the which breaks down the cell membranes

Detergents are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranes

The end result of LYSIS is that the contents of the plant cells are distributed in solution.

PRECIPITATION : THIS A SERIES OF STEPS WHERE DNA IS SEPARATED FROM THE REST OF THE CELLULAR COMPONENTS

In a research lab, the first part of precipitation uses phenol/chloroform to remove the proteins from the DNA

Phenol denatures proteins and dissolves denatured proteins. Chloroform is also a protein denaturant

The second part of research lab DNA precipitation is the addition of

salts The salts interrupt the hydrogen bonds between the water and

DNA molecules.

The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol

In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution.

The DNA is pelleted by spinning with a centrifuge and the supernatant removed.

Washing: The precipitated DNA is laden with acetate

salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA.

Resuspension: The clean DNA is now resuspended in a buffer

to ensure stability and long term storage. The most commonly used buffer for

resuspension is called 1xTE(The purpose of TE buffer is to solubilize DNA or

RNA, while protecting it from degradation) EDTA further inactivates nucleases, by binding to metal cations required by these enzymes

Washing and Resuspension:

CHECKING THE QUALITY OF YOUR DNA The product of your DNA extraction will be

used in subsequent experiments Poor quality DNA will not perform well in PCR You will want to assess the quality of your DNA

extraction using the following simple protocol: Mix 10 µL of DNA with 10 µL of loading buffer Load this mixture into a 1% agarose gel Analyze results (the following slides provide

guidance)

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METHODS OF SEPARATING DNA

Polyacrylamide gel electrophoresis 20bp - 2000bp

Conventional agarose gel electrophoresis 300bp - 40,000bp 100bp-2000bp (special agaroses) low melting point agaroses

Pulse field/CHEF 40kbp - 2000kbp

If properly done, genomic extraction should result in bright bands in the very high base pair range of a gel electrophoresis.

Sizes of Genomic DNA for various Species in kbp

E. Coli 4,640,000bp

Yeast 12,100,000bp

Fruit Fly 140,000,000bp

Human 3,000,000,000bp

Pea 4,800,000,000bp

Wheat 17,000,000,000bp

The genomic fragments run at ~12kbp because they are sheared during extraction

Analyzing DNA Samplesin a Research Lab

Nucleic Acid Analysis via UV Spectrophotometry

By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak at ~260nm.

[dsDNA] ≈ A260 x (50 µg/mL)[ssDNA] ≈ A260 x (33 µg/mL)[ssRNA] ≈ A260 x (40 µg/mL)

DNA Absorption Spectra

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