Bio 230 Genetics

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Markers tell us if the cells have been transformed

Both the vector and the (other DNA) have to be cut with the same the restriction enzyme

Self-ligation – vector without the new DNA from the other organism.

Competent cells- tortured (heated to a temperature that they barely survive) which loosens the membrane so that the vector is more likely to be accepted

Inside the vector a gene encodes for antibiotic resistance. Only antibiotic resistance get with the plasmid. The lacZ gene can be expressed into an active protein which can cut x-gal (colourless). As soon as x-gal is cut it turns into a blue product.

Colony selected: x-gel = white as a blue indicates a “broken” lacZ

As DNA -> mRNA -> protein it instead becomes D—NA ->MR---NA -> pro---tein

Vectors:

Plasmids can only take an insert or 25 kbs (bases)

Expression vectors – turns on gene of interest

Ti vector- only works for plants used for GMO

Host cells:

Saccharomyces cerevisiae – used for vaccines and flavoring

Transgenic plants – see slides

Ethics – want labels in Europe, North America – don’t care

Golden rice – increases nutritional value – (Vitamin A – lack of causes blindness and death) – not commercialized – seeds can reproduce.

How to distinguish transgenic plants from wild?

Isolate the entire DNA then analyze the DNA. Detected by PCR - polymerase chain reaction

PCR

1st step – denaturing – over 90 C

2nd step – annealing – primers (short sequences) bind to the DNA – 20 to 25 nucleotides long. Forward primers on one strand and the reverse primer on the other strand. If primers are 100% match the temperature can be 60 C if less then the temperature should be around 40 C.

3rd step – elongation – add all the building blocks (dNTPs) primers are start signal followed by the building blocks. Happens at 72 C.

Every cycle doubles the original number.

The whole point is to get a specific strand of DNA – multiple strands in order to analyze, sequence etc.

PCR – developed in the 80’s and received an nobel prize in the 90’s

Limitations of PCR – must have sequence information

- Cannot amplify a sequence (sequence endlessly)

- Must wear gloves – easy to contaminate the sequence

Applications of PCR – amplify a specific sequence

- Genetic screening – using specific primers

- Blood testing – diagnose

- Paternal testing

Real time PCR - detects gene expression

Visualize DNA (or RNA) – agarose gel electrophoresis

Large molecules move slowly and small molecules move fast. Add electricity, the negatively charged DNA moves towards the + anode. Add cyto-green so under UV light you see the bands.

If the gel has a continuous blob – the bands are super close together

Foreign DNA can be expressed because of the genetic code is universal

Gene Transcription

The genetic code is linear – a sequence of nucleotide bases (doesn’t branch)

Triplet code: 3 nucleotides=1 protein

Unambiguous: only encodes for 1 amino acid

Degenerate: amino acid 1 can be encode by a triple code A or triple code B

Start and stop codons: ATG = start codon (M) and three stop codons (do not encode for an amino acid)

Commaless: Unless stream of nucleotides, no idea where genes start and end

Nonoverlapping: A base can only encode towards one amino acid

Colinear: Linear DNA = linear RNA = linear protein sequence

Universal: The genetic code is universal – all organisms use the same genetic code

In the 50s Volkin worked with bacteriophages to indicated that DNA is the template.

Four nucleotides can be translated into 20 amino acids by:

- Mathematical: 43 = 64 – Crick uses this theory to discover that DNA is read in triplets, exposed to a chemical that causes a mutation. Once a nucleotide is added it creates a completely different sequence.

- Deleting 1 or 2 nucleotides alters the entire strand but deleting 3 nucleotides just an amino acid is missing (which could still allow the protein to be functional)

Check for frame shift mutations on the exam!!!

Brenner discovered that if overlapping would only allow 16 combinations to precede or follow – limiting the number of amino acids that can follow each other.

- The effect of a single nucleotide change: if overlapping existed, a single base change would change multiple amino acids.

Adaptor molecule = mRNA = Non overlapping

In vitro protein – synthesizing system

- Out of the cell – controlled environment

- mRNA created without DNA by using polynucleotide phosphorylase (enzyme)

- Usually this enzyme = breaks down into individual units (degrades) when added in excess it forces the synthesis of RNA

RNA