Micro 10 Transcription and Translation

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Transcription DNA -> RNA the 1st step to making

protein.

The single strand of RNA that results from reading the DNA is called

messenger RNA (mRNA)

1st step RNA polymerase binds to a promoter

is just a codon that says "start here"

T T C G A G C A

DNA

A A G C U C G U

RNA

Transcription RNA polymerase binds to the

promoter

RNA Transcription, Moves down the strand of

mRNA reading each base and building RNA's complimentary base.

RNA Transcription Arrives at a STOP signal and

detaches.

RNA Transcription

Result is a single strand of mRNA

Translation mRNA ->

protein

Translation, Ribosomal RNA (rRNA) binds to the

mRNA

Ribosomal RNA

rRNA

Translation, The rRNA "translates" the mRNA into an

amino acid sequence

The entire process of transcription and translation is are referred to as

gene expression.

Gene expression, Every gene ultimately results in the production of

protein.

Phenotype

The physical appearance or functional expression of a trait. (blonde hair, green eyes, estrogen receptor)

Genotype

The genetic code underlying a single trait or set of traits. (specific DNA sequence for blonde hair, green eyes, estrogen receptor)

Regulating Gene Expression, How are transcription and translation controlled

60-80% of genes are constitutive

60-80% of genes are constitutive and

Produced at a fixed rate

How are transcription and translation controlled

Two mechanisms control gene expression Repression & Induction

Two mechanisms control gene expression

Repression & Induction

Repression Inhibits

Transcription

Repression is Usually due to a

build up of end-product

Repression , As the end-product builds up, repressors bind to the

promoter region of DNA and prevent RNA polymerase from binding

Transcription is turned on by an

inducer.

Enzymes that require an inducer to be produced are called

inducible enzymes

Lactose acts as an inducer for

b-galactosidease (ONPG)

b-galactosidease breaks lactose into

glucose and galactose.

Mutations are changes in the

hereditary message of an organism.

Two categories of mutations

Sequence changes & Changes in Gene position

Sequence changes

Base substitution, Chemical modification, DNA breaks, Slipped mispairing , Triplet expansion

Changes in Gene position

Chromosomal rearrangement & Insertional inactivation

Sequence Changes

Base substitution, Normal C G T C A, Base sub C G G C A, Occurs during replication, Also called a point mutation

Sequence Changes

Chemical modification, A base becomes chemically modified by a mutagenic chemical, Another class of point mutation

Sequence changes DNA

breaks

Sequence changes Caused by

Ionizing radiation

Sequence changes Causes

double strand breaks

Sequence changes Results in the

loss (deletion) of short segments

Sequence changes Slipped

Mispairing

Sequence changes Results in

a deletion

Sequence changes Also called a

frameshift mutation

Sequence changes, A sequence is present in

more than one place on a strand and pairs out of order

Sequence changes Like a shirt buttoned on the

wrong button

Triplet Expansion is When a

3-base sequence is repeated several times in a gene.

Triplet Expansion Results in

mutant proteins

More than 15 human diseases are attributed to

triplet expansion

Mutations - Changes in Gene Position

Chromosomal Rearrangement

Translocation

Part of one chromosome becomes a part of another chromosome

Inversion

Orientation of a portion of a chromosome is reversed

Duplication (gene abnormalities)

Entire regions of chromosome become duplicated

Deletion(gene abnormalities)

Similar to the deletion on a nucleotide level but much larger amounts of DNA are deleted

Aneuploidy (gene abnormalities)

Whole chromosomes are lost or gained

Polyploidy(gene abnormalities)

An entire SET of chromosomes are added.

Genetic Recombination Technology, Modern scientists can insert or delete gene segments to

form new combinations of genes

Genetic Recombination Technology

Genes are usually inserted

Genetic Recombination Technology, Vaccine development is trying to delete

pathogenic genes.

What do the letters PCR stand for

Polymerase, Chain, Reaction

PCR is the amplification of a

DNA sequence

PCR is the name of the assay that scientists use to look at what genes are being expressed in a

cell, tissue, or organism.

Why amplification? We have not yet developed the technology to

detect 1 copy of DNA although it is fast approaching.

Orientation to DNA, DNA is made up of

4 nucleotides

DNA , 4 nucleotides 1

A - Adenine

DNA , 4 nucleotides 2

T - Thymine

DNA , 4 nucleotides 3

C - Cytosine

DNA , 4 nucleotides 4

G - Guanine

A =

T ALWAYS

C=

G ALWAYS

DNA Replications Major Players

DNA, DNA polymerase, Nucleotide Bases (A's, T's, C's G's)

DNA Polymerase...what is it? It is the enzyme in your body responsible for

naturally replicating your DNA

DNA Polymerase...what is it? It is the most accurate enzyme making less than

one mistake for every billion bases

DNA Polymerase...what is it? It has a built in

proofreading capability

DNA Replication, Where to start and stop? Mother nature uses

"codons"

DNA Replication, Where to start and stop? Scientists use

primers

Primers let scientists designate where they would like

amplification to begin and end.

What is PCR?

The amplification of a DNA sequence

What is in a PCR tube?

DNA, Primers, A's T's C's and G's, DNA Polymerase

PCR reaction, PCR is broken into

cycles

PCR reaction 94°C for 1 min

denaturation

PCR reaction 55°C for 20sec

annealing

PCR reaction 72°C for 2min

elongation

Taq Polymerase The reason that

PCR is possible

Taq Polymerase was Found in the bacteria that exist in

hot springs

PCR Applications, Why would you ever need to amplify the whole genome?

Forensics, Archeology, Disease Diagnosis

Who Pioneered PCR?

Dr Kary B. Mullis

Dr Kary B. Mullis Won the

Nobel Prize for Chemistry in 1993.

PCR in Research Scientists rarely have a need to look at

the whole genome

PCR in Research Scientists want to look at expression of

specific genes.

How are specific genes expressed? Does every cell carry the entire genetic code?

YES

How are specific genes expressed? Does every cell express the entire genetic code?

NO

(RT-PCR) What does the RT stand for?

Reverse Transcription

Reverse Transcription (RT-PCR)How?

Reverse Transcriptase

Reverse Transcriptase

Retro-viruses have it

Reverse Transcriptase VERY

sloppy enzyme, 1 mistake in every 2,000 bases

Inside an RT-PCR tube Ingredients

mRNA, Primers, Taq, Reverse Transcriptase, dNTP's

Reverse Transcriptase, Primers, and dNTP's make the first strand of

cDNA.

After the first strand of cDNA is made the other primer binds and Taq helps to make the first

dsDNA

After the creation of dsDNA, PCR continues on as before

Denaturing, Annealing, elongation

(Primers) It is generally accepted that if you find "part" of the message you have found

the entire message

(Analyzing RT-PCR)What is Gel Electrophoresis?

Forcing molecules through a gel with electric charge.

Analyzing RT-PCR Big/Heavy molecules vs

Small/Light molecules.

What charge does DNA carry?

Negative

How do we see DNA in a gel?

Ethidium Bromide

Ethidium Bromide, How does it work?

Intercalating agent.......Fluoresces under UV light

Analyzing a gel

DNA bp ladder, Positive Control, Negative Control, Samples

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