biology chapter 21 manipulating genomes

introns - non coding DNA

exons - coding DNA

what are introns and exons?

short DNA sequences repeated many times

satellite DNA?

minisatellite: 20-50 base pairs repeated 50-100

microsatellite: 2-4 base pairs repeat 5-15 times

Different people have different numbers of repeats so different satellite patterns which is the basis of DNA profiling

minisatellite and microsatellite definition?

1. put DNA fragment through PCR (polymerase chain reaction)

2. restriction endonucleases are used to cut into small fragments at restriction sites

3. put onto gel and do gel electrophoresis. DNA are attracted to the positive charge and large strands move further

4. add a radioactive or flourescent dye to see evidence

how to produce a DNA profile?

1. denaturation: 95 degrees to break hydrogen bonds in DNA

2. Annealing of primers at 55 degrees. They bind to the end of the DNA strands

3. At 72 degrees, synthesis of a new DNA strand with free nucleotides occurs. Done by TAQ polymerase (obtained from thermophilic bacteria in hot springs)

steps for PCR

• crime scene DNA and forensics

• paternity tests

• analysis of risk of diseases

• evolutionary relationships between species

uses of DNA profiling

• developed by Sanger and paved the way towards new developments

• Needs a DNA sample, free nucleotides, coloured terminator bases, DNA polymerase and primer to start process

what is needed for sequencing DNA?

1. heat up to 95 to denature and break hydrogen bonds

2. cool to 50 and the primers will anneal

3. heat up to 60 and DNA polymerase starts adding free nucleotides bases

4. a terminator base is added and since they are missing an oxygen, they can’t form phosphodiester bonds and will stop base addition. This creates a variety of lengths of DNA.

5. After gel electrophoresis, direct UV light at nylon to see the lengths.

6. Can also do capillary sequencing and read with a laser to construct the genome

how to do DNA sequencing

• fragments of DNA attached to the slide called a flow cell and replicated using PCR to form clusters

• all clusters sequenced at the same time so it is cheaper and faster

Next generation sequencing/ high throughput sequencing

software needed to organise and analyse raw biological data

What is bioinformatics?

uses data from bioinformatics to build theoretical models of biological systems

what is computational biology?

• analysing the genomes of pathogens to find the source of infections and the medicine needed to fight it

• Identifying species with DNA barcoding

• allowed us to find the sequence of amino acids in a polypeptide to be predicted

How is DNA sequencing used?

design and construction of new artificial biological pathways

genetic engineering, making new genes to replace faulty ones, synthesis of whole new organisms, use of biology industrial contexts

what is synthetic biology and some techniques?

1. human cell DNA is extracted out and plasmid is removed from bacteria using endonucleases or reverse transcriptase

2. restriction endonucleases are used to cut out desired gene and same enzyme used to cut plasmid which leaves same sticky ends. DNA ligase forms bonds between gene and plasmid. This is called a recombinant plasmid.

3. Recombinant plasmid put into bacteria now called transgenic bacterium.

4. Put onto agar plate to culture it.

Genetic engineering method

they have a marker gene which is a gene that has been engineered into the plasmid and enables the scientists to see which bacteria have taken the plasmid.

Why are specific plasmids or vectors used in genetic engineering?

to show that the plasmid has the recombinant gene

if inserted successfully, marker gene will not function like the flourescence gene

what is the second marker gene used for?

1. culture in Ca2+ solution and heat shock which causes membrane to become permeable

2. electroporation to create pores with electricity

3. Electrofusion to fuse cells and create a polyploid cell with DNA from two cells fused

how is the plasmid with recombinant DNA put into the host cell?

• mutant allele is replaced with healthy allele in somatic cells (body)

• temporary solution and disease can still be passed onto offspring

what is somatic cell gene therapy?

• insert healthy allele in eggs or embryo cells after fertilisation

• healthy allele would be passed onto offspring

• done on animals but not humans as it is done to embryos without consent

what is germ line cell gene therapy?

people were initially uncomforatble with using human genes in microorganisms but the human medicines they make are very useful.

ethical concerns of using GM bacteria?

benefits:

• reduce pesticides used

• increased yield

• disease resistant

risks:

• non pest insects might be damaged by toxins in GM plants made for pests

• transferred genes for disease resistance may spread to the wild populations

• biodiversity reduced when herbicides used to kill weeds

benefits and risks of GM crops?

• swine fever-resistant pigs

• Faster-growing salmon

• Pharming:

  • creating animal models so animals develop certain diseases to be tested on

  • creating human proteons

ethical issues as they cannot give consent and could cause them harm>

how have animals been GM’d?

ethical issues?