manipulating genomes

What is Sanger sequencing

Chain termination method

Chain termination method of DNA uses modified nucleotides called what?

Dideoxynucleotides - they pair with nucleotides on template strand during DNA replication

How does chain termination reaction work

4 test tubes are prepared that contain the DNA to be sequenced

Test tubes are incubated

The primer anneals produce a short section of double stranded DNA at the start of the sequence

DNA polymerase attaches to double stranded section and begins DNA replication using free nucleotides

Hydrogen form between complementary bases

At any time DNA polymerase can insert one of dideoxynucleotides which results in termination of DNA replication

Once incubation period has ended, new complementary DNA chains are separated from template DNA.

This results in single stranded DNA

What is high throughput sequencing?

Simultaneous sequencing of multiple DNA strands and is usually rapid

also called next generation sequencing

What is bioinformatics and how has it helped genome comparisons

Software developed to process and understand large complex data

It allows access to large amounts of data so comparisons can be made with genomes of an organism

Information is universal

amino acid/protein structures held in database

Allows rapid comparison of sequences with alllesa

How do genetic variation and evolutionary relationships allow comparison

Genetic variation can be investigated by - species that have a high genetic variation will exhibit a large number of differences in base sequences

For evolutionary relationships genome of species can be compared by: seeing if species share a common ancestor and the protein cytochrome c in respiration

How does genotype phenotype relationships allow comparison

Scientists “knock out” (stop expression) of genes. When genome sequence is known scientists target base sequence to knock out

What is epidemiology and how is bioinformatics used in it

Study spread of infectious disease within populations

Pathogens are analysed to aid in research and disease control

Bioinformatics allows:

Highly infectious strains to be identified

The ability of pathogens to infect multiple humans is identified

Can design vaccine programmes

information generated from HGP

Finding cures for diseases

Changes in specific genes and likelihood of developing certain inherited diseases

How has gene sequencing allowed the development of synthetic biology

Synthetic biology involves redesigning organisms for useful purposes by engineering them, assembly of a new genome can be done using existing DNA sequences.

Requirements for PCR

Target DNA or RNA being amplified

Primers

DNA polymerase

Free nucleotides

Buffer solution

What is PCR used for

Produce large quantities of specific fragments of DNA and RNA

Key stages of PCR

Denaturation - double stranded DNA is heated to 95° which breaks hydrogen bonds that bond the two DNA strands together

Annealing - the temperature is decreased to between 50-60° so that primers can anneal to the ends of single strands of DNA.

Elongation / Extension - the temperature is increased to 72° for at least a minute as this is the optimum temperature for Taq polymerase to build complementary strands of DNA to produce new identical double stranded DNA molecules

Electrophoresis is used to analyse what

DNA, RNA, proteins

What are restriction endonuclease enzymes

Cut DNA into fragments

What is VNTR

Variable number tandem repeats - they are found on non coding parts of DNA

During electrophoresis the molecules are separated according to their what?

Size/mass - different sized molecules move through gel at different rates

, type of gel -different gels have different sized pores that affect speed what molecules can move through

electrical charge - positive molecules move to cathode, negative molecules move to anode, DNA is negatively charged so move to anode

What is the method for gel electrophoresis

Create an agarose gel plate in a tank, wells are cut into gel at one end

Submerge the gel in an electrolyte solution in the tank

Load fragments into wells using a micro pipette

Apply an electrical current to the tank, DNA current moves to anode (positive electrode) because DNA is negatively charged

The smaller mass/fragments will move faster and further from wells

The fragments are not visible so must be transferred onto absorbent paper,or nitrocellulose where probes eg: radioactive label or fluorescent stain are added

What are probes

Single stranded DNA sequences complementary to VNTR regions

DNA profiling helps with what

Identify suspects for crimes and identify corpses because everyone has a unique VNTR (variable number tandem region

Can also be used for d to identify people at risk of diseases

Determine familial relationships (eg: who your father may be)

Used in species conservations

How does DNA profiling work?

What are the steps

DNA (indicated by bars) creates a Profile or fingerprint

1.DNA is obtained through hair, blood, semen, saliva

2.Increase quantity of DNA using PCR

3Use restriction endonuclease to cut amplified DNA

4.Separate fragments using gel electrophoresis

5.use radioactive or fluorescent probes that are complementary to bind to VNTR

6.X-ray images are produced or UV light is used to produce images

7. These images contain patterns of bars (DNA profile) which is then analysed

What is genetic engineering

Manipulation of DNA sequences of an organism

Scientists have been able to change an organisms DNA by doing what

Combining lengths of nucleotides from different species

Altered DNA with introduction of nucleotides is called what

Recombinant DNA (rDNA)

If an organism has a nucleotide sequence from a different species it is called what

Transgenic organism

An organism with genetically introduced genetic material is called what

Genetically modified organism

How does Recombinant DNA in technology work

Transfer fragments of DNA from one organism/species into another

This organism will contain recombinant DNA and be a genetically modified organism

Steps of genetic engineering

Identification of a DNA fragment or gene

Isolation of the desired DNA fragment (using restriction enzyme or reverse transcriptase)

Multiplication of DNA fragment using PCR

Transfer into organism using a vector

Identification of cells with a DNA marker

Tools needed to modify an organism

Enzymes: restriction endonuclease- cut genes at specific base sequences. Ligase- join cut ends of DNA to form phosphodiester bonds. Reverse transcriptase - build up double stranded DNA from single stranded RNA

Vectors (transfer fragments into cell:

Plasmids - transfer DNA into bacteria or yeast

Viruses - transfer DNA into human cells or bacteria

Liposomes + fuse with cell membranes to transfer DNA into cells

Markers -(genes that code for identifiable substances)

Fluorescent markers, enzyme markers, antibiotic resistance markers

Uses of genetic engineering

Genetically modify crops to make them resistant to drought and disease

Genetic modification of livestock to make them disease resistant

Genetic modification of bacteria to produce medicines eg: insulin

Other Uses of genetic engineering

Used in microorganisms to produce proteins eg: bacteria yeast and animals cells for treatments. This is good as: it is simple, it’s fast, reliable supply, proteins are engineered to be the same as human proteins

Used in human insulin production. This is good as: bacterial insulin is identical to human insulin, reliable supply, cheaper, fewer ethical, moral, or religious concerns

Animals - to get desired characteristics faster, all organisms contain desired characteristics or to grow faster

Soya - to release toxins to kill insects

Pathogens for medical treatments.

Arguments against genetically modified organisms

Biotech companies charge farmers more money for GM seeds

Many people object to food production in GMOS as there is lack of long term research

Environmentalists concerned about reduction in biodiversity

Resistant herbicides can transfer into super weeds

Pollen from GM crops contaminate other GM crops

Superbugs can arise from antibiotic resistant pathogens

Mutations may occur in inserted genes

Negatives on genetically modified pathogens for research

Health and safety risks of the researcher and the wider public

What is Pharming

Genetic modification of animals

Ethical concerns for Pharming

Is it right to put human genes into Animals?

Welfare of animal could be at risk

Creating transgenic animals might cause harm to animal

2 types of somatic gene therapy

In vivo - the gene is inserted via a vector into cells inside the body

Ex vivo - the gene is inserted via a virus vector into cells outside the body