1) Infection Immunology and Forensics

8/2/2019 1) Infection Immunology and Forensics

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DNA carries all genetic information.

Double helix structure containing chains of nucleotide bases, A, G, T, C:

Adenine Thymine

Guanine

Cytosine

Each living organism is unique and has different genetic message.

Sequence of bases are a genetic code and determine the amino acids proteins.

Proteins and enzymes control synthesis and biochemistry of cells and organism.

Proteins are formed from amino acids.

20 naturally occurring amino acids.

They join to make combinations on the surface of the ribosome's.

The order they join is due to the genetic code.

DNA is a double helix.

Triplet code . 4 x 4 x 4 = 64 possible combinations.

A sequence of three bases on DNA is transcribed to give the

Corresponding base pair ofmRNA (messenger RNA) in the nucleus.

Part of Protein Synthesis


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CONDENSATION REACTION

PHOSPHODIESTER BOND =

The process repeats so that a very long chain

of nucleotides is made, a polynucleotide

There will be a spare 5' sugar atom at one endof the chain and a spare 3' atom at the other.

The chain thus has a 3' to 5' direction reading

up the page.

In DNA a second polynucleotide chain forms

next to the first, but this runs in the opposite

direction. The chains are therefore describedas antiparallel.


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The bases now find themselves opposite one another and bond together with weak hydrogen

bonds. When this occurs Adenine always pairs with Thymine (A-T) and Guanine with Cytosine

(G-C). There is a good reason for this complementary pairing.

Adenine andGuanine both have

a double ring

structure and are

classified chemically

as Purine bases.

Thymine, Uracil and

Cytosine all have a

single ring structure

and are classified as

Pyrimidines.


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Degenerate Code

There is a lot of degenerate or redundant code.

Often only the first 2 nucleotides code for theamino acid.

UUU

UUC both code for phenylalanine despite

having a different last nucleotide.

This is good as if amino acids where caused by just

one codon then a mutation would have a huge

effect.

If a mutation happens on the last base then it will

still produce the same amino acid.

64 combinations but some code for the sameamino acid:

Genetic Code is Non-overlapping and Degenerate

Non-Overlapping

An overlapping code would be very economical as relatively short lengths of DNA would be

needed to code for many different proteins.

However this would be very limiting as the amino acids which where side by side would belimited.

For example only leucine would ever be able to follow phenylalanine.


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DNA is in the nucleus.

Proteins are made on the ribosome's.

The message of DNA has to be copied to form a messenger RNA (mRNA).

Break down of hydrogen bonds by RNA polymerase.

Only the 5 prime DNA strand is transcribed to give a single strand of mRNA.

This is known as template strand as it provides the template for ordering the sequence of

nucleotides in an RNA transcript.

Transcription also uses DNA polymerase.

Enzymes join many nucleotide units together to form mRNA.

Each triplet code produces a Complementary Codonon the mRNA.

The available region of DNA template is read 3-5 by the RNA polymerase, so the new RNA is

synthesised 5 3.

Thymine is replaced by Uracil in RNA.

The new RNA strand has the same sequence as the

Non template strand.

This is then known as the sense strand (the same).

The template strand as the antisense strand (opposite).


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Ribosome structure:

Large sub unit

Small sub unit

Histone Proteins

Ribosomal RNA

Made in the nucleus

then moves out when

made.

RNA structure

Made in cytoplasm Unit of 3 bases at one end known as

anticodon.

mRNA recognition end

These pick up amino acids

Each amino acid has its own tRNA

molecule.


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There are three RNA: mRNA, tRNA, ribosomal RNA.

Ribosomes surround the mRNA being translated.

The sub units hold the mRNA, tRNA and enzymes together controlling the protein synthesis.

Each strand has a start codon (AUG) and a stop codon.

When the mRNA attaches to the ribosome it starts reading at the start codon.

After this AUG stands for an amino acid. Molecules oftRNA carry amino acid to the surface of the ribosome.

tRNA lines up its anticodons alongside a complementary codon.

Hydrogen bonds bind tRNA in place while enzymes link amino acids in peptide bonds.

tRNA then returns to cytoplasm to pick up another amino acid.

Ribosome moves along revealing new codons.

Until the stop codon.


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Actually after unravelling haemoglobin molecule they realised they where made up of many

polypeptide chains and different mutations changed different strands.

SO One Gene One polypeptide.

So each gene is transcribed to give a strand of mRNA which is translated to produce a uniquepolypeptide sequence.

RNA transcribed from DNA is now referred to as pre-mRNA

It contains RNA copied from all the DNA in the gene.

Including nonsense sections which dont code for proteins.

Introns nonsense sections of DNA/pre-RNA.

Exons rest of RNA is a copy of the areas of DNA that do code for the polypeptide.

When the mRNA (pre mRNA) is first transcribed it is not yet finished.

A number of modifications take place:

Capping of the end of the strands so it isn't attacked by enzymes.

And THE REMOVAL OF INTRONS

The remaining Exons are joined together after the RNA splicing .

Spliceosomes are the enzymes that do this.

These are all post transcriptional changes which lead to more variety in phenotypes.

Some exons are sometimes also removed so RNA is different to DNA and more codes

possible: So 25000 genes code for 90000 different proteins.


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You can identify organisms and people by their sequences of DNA.

DNA profiling

Human genome = 20 000 25 000 genes and chromosomes are made up ofmillions of base

pairs.

Less than 2% codes for proteins. 90% is introns and repeats.

Introns are not yet known for purpose but can be used for profiling.

Within introns there are short sequences of DNA that are repeated many times:

Mini-Satellite20-50 bases repeated 50 to several hundred times.Micro-satellite2-4 bases repeated 5-15 times.

The same mini and micro satellite appears in the same position on each homologous pair of

chromosomes.

The number of repeats however vary as different repeats are inherited.

These repeats are known as SHORT TANDEM REPEATS

There are many different introns and a huge variety of repeats so the likely hood of any two

individuals having the same pattern of DNA is very unlikely making everyone so unique.


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Strands of DNA are chopped into fragments with restriction endonucleases

These enzymes cut the DNA at particular points into intron sequences.

Each enzyme cuts at different points known as recognition points.

Leaving micro and mini satellite sequences.

The fragments are then separated and identified.

GEL ELECTROPHORESIS: (chromatography).

DNA fragments placed in wells in an agarose gel medium in a buffering solution and known

DNA fragments.

The Gel Contains a dye which under UV shows DNA bands.

The DNA also contains a dye that moves faster than DNA through gel to show when to stop

experiment.

Electric current is passed through the apparatus and the DNA fragments move towards

positive as negative charge on phosphate.

Move at different rates depending on mass and charge.

Placed under UV

DNA fluoresces in bands to be identified.

It shows large DNA fragments containing

50 base pairs (mini satellites).


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Southern Blotting:

An alkaline buffer solution is added to

the gel after electrophoresis and a nylon

filter or nitrocellulose paper.

Dry absorbent paper is used to draw thesolution containing the DNA fragments

from the gel to the filter leaving blots.

The alkali solution also separates out the

fragments so individual bases can be

seen.


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1) Infection Immunology and Forensics