Dna in basic

DNA: The Secret Of Our Life(An Introduction Lecture)

lecture 1

Lecture Objectives

• At the of this Lecture you should be able to learn:

• 1. The history of DNA discovery with their importance for the new concepts and application techniques

• 2. The chemical and physical properties of DNA and how these properties were exploited for DNA technologies

Early historical perspective

We enter the 20th century with an understanding of the DNA building

block.

Some Experimental Data leading to DNA as biological source of DNA

• Griffith’s • Avery et al.

• Hershey and Chase

• Chargaff

• Wilkins and Franklin

• Watson and Crick

Griffith’s Experiment: 1928

Conclusion: A Transformation “factor” exists

Support for nucleic acid transfer

Hershey and Chase Experiment, 1952: Confirms DNA as genetic material

Conclusion: DNA identified as source of genetic information

Franklin and Wilkins 1947

1920-1958 1916-2004

Chargaff’s Rule 1948

1905-2002 1905-2002 1905-2002

1905-2002

Watson and Crick, 1953 inferred the DNA structure

Nobel Prize: 1962

1928-1916-2004 1916-2004

The building blocks of DNA are nucleotides.

RNA’s Sugar DNA’s Sugar

Nitrogenous Bases

DNA nucleotides

Polarity and Anti-Parallel

Back to Franklin and Wilkins Data: Pairing of specific classes of bases can account for diameter of DNA

Just right!

6 sided ring

6 sided ring + 5 sided ring

Most Common Secondary Structure (3D structure)

• B-DNA

• Alpha Helix

• Right Handed Turn

• 10 bases per 360º turn

A function of Major and Minor Grooves

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Nucleosides• Nucleosides: nitrogenous base linked to specific sugar

– RNA: adenosine, guanosine, cytidine, uridine– DNA: deoxyadenosine, deoxyguanosine, deoxycytidine,

(deoxy)thymidine

138.192.68.68/.../Nucleosides.gif

DNA nucleoside RNA nucleoside

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Nucleotides

The nucleotide structure consists of– the nitrogenous base attached to the 1’ carbon

of deoxyribose– the phosphate group attached to the 5’ carbon

of deoxyribose– a free hydroxyl group (-OH) at the 3’ carbon of

deoxyribose

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Nucleotides

• Subunits of DNA and RNA– Nucleosides

linked to phosphate group via ester bond

– “dNTP’s”: DNA– “rNTP’s”: RNA

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DNA Structure

Nucleotides are connected to each other to form a long chain

phosphodiester bond: bond between adjacent nucleotides– formed between the phosphate group of one

nucleotide and the 3’ –OH of the next nucleotide

The chain of nucleotides has a 5’ to 3’ orientation.

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DNA structure determinationChargaff's Rules

– Erwin Chargaff determined that • amount of adenine = amount of thymine

• amount of cytosine = amount of guanine

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DNA StructureThe double helix consists of:

– 2 sugar-phosphate backbones– nitrogenous bases toward the interior of the

molecule– bases form hydrogen bonds with complementary

bases on the opposite sugar-phosphate backbone• Adenine pairs with Thymine (2 H bonds)• Cytosine pairs with Guanine (3 H Bonds)

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DNA Structure

The two strands of nucleotides are antiparallel to each other– one is oriented 5’ to 3’, the other 3’ to 5’

The two strands wrap around each other to create the helical shape of the molecule.

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Chemical Properties of DNA

• Factors that affect DNA structure– Temperature: denaturation (can be reversible)– pH: high pH can denature DNA– Salt concentration: lowering salt concentration

can denature DNA– Molecular Hybridization (DNA:DNA) and

(DNA:RNA)– UV absorption (230-260nm)

• Southern blotting of DNA fragmentsAPPLICATION Researchers can detect specific nucleotide sequences within a DNA sample with this method. In

particular, Southern blotting is useful for comparing the restriction fragments produced from different samples of genomic DNA.

TECHNIQUE In this example, we compare genomic DNA samples from three individuals: a homozygote for the normal -globin allele (I), a homozygote for the mutant sickle-cell allele (II), and a heterozygote (III).

DNA + restriction enzyme Restrictionfragments I II III

I Normal-globinallele

II Sickle-cellallele

III Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

Gel

Sponge

Alkalinesolution

Nitrocellulosepaper (blot)

Heavyweight

Papertowels

1 2 3

Figure 20.10

RESULTS Because the band patterns for the three samples are clearly different, this method can be used to identify heterozygous carriers of the sickle-cell allele (III), as well as those with the disease, who have two mutant alleles (II), and unaffected individuals, who have two normal alleles (I). The band patterns for samples I and II resemble those observed for the purified normal and mutant alleles, respectively, seen in Figure 20.9b. The band pattern for the sample from the heterozygote (III) is a combination of the patterns for the two homozygotes (I and II).

Radioactivelylabeled probefor -globingene is addedto solution ina plastic bag

Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globinallele

Paper blot

Film overpaper blot

Hybridization with radioactive probe. Autoradiography.

I II IIII II III

1 2

• DNA microarray assay of gene expression levelsAPPLICATION

TECHNIQUE

Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNAmicroarray

Size of an actualDNA microarraywith all the genesof yeast (6,400spots)

Isolate mRNA.1

With this method, researchers can test thousands of genes simultaneously to determine which ones are expressed in a particular tissue, under different environmental conditions in various disease states, or at different developmental stages. They can also look for coordinated gene expression.

Make cDNA by reverse transcription, using fluores-cently labeled nucleotides.2

Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism‘s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.

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Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample.

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RESULT The intensity of fluorescence at each spot is a measure of the expression of the gene represented by that spot in the tissue sample. Commonly, two different samples are tested together by labeling the cDNAs prepared from each sample with a differently colored fluorescence label. The resulting color at a spot reveals the relative levels of expression of a particular gene in the two samples, which may be from different tissues or the same tissue under different conditions.

Figure 20.14

Central Dogma

Information Transfer