Application of genetics!!

Define donor DNA

- a gene isolated for insertion

Define plasmid

- circular loops of DNA found in bacteria and used as a vector

Define restriction enzymes

- enzymes that cut DNA molecules between specific base sequences

Name the enzymes that joins together portions of DNA

- DNA ligase

What are sticky ends in DNA?

- the 2 ends of the foreign DNA segment

- they have a short row of unpaired bases that match the complementary bases at the 2 ends of the opened-up plasmid

Define recombinant DNA

- DNA which results from the combination of fragments to different organisms

Define reverse transcriptase

- enzymes used to synthesis DNA from mRNA in specific cells

state uses of genetic engineering

- to transfer genes into bacteria

- to transfer genes into plants and animals

- to transfer genes into humans

state stages of genetic engineering

- isolation of DNA fragment including the gene required

- the incorporation of DNA fragments into a vector

- the introduction of the vector into the host cell

- the identification of the host cells that have taken up the gene by use of genetic markers

- replication of the host cells

state different ways to isolate the gene rom the donor DNA molecule

- mRNA copy of the gene can be used to make DNA using reverse transcriptase

- the required gene can be located on the chromosome and restriction endonucleases used to cut out the gene

- the protein required can be sequenced and then the base code can be found and DNA made

outline how you would isolate the required gene from donor DNA using mRNA version

- mRNA sample would be in a cell expected to have high quantities of mRNA (eg: cytoplasm of pancreatic cells have mRNA coding for insulin)

- extract mRNA

- use reverse transcriptase (from retrovirus) to synthesis DNA sequence from mRNA

- DNA produced will be single stranded and called cDNA (copy DNA)

- DNA polymerase converts this to a double strand for incorporation into a plasmid

outline how you would isolate the required gene using by cutting it out

- use DNA probe to locate specific gene coding for protein

- the target gene is isolated using restriction endonucleases. - The enzyme cuts DNA between specific base sequences

- can either be cut straight but most restriction enzymes cut staggered leaving unpaired bases on both strands (sticky ends)

which ways of isolating desired gene requires sicky ends

all

Disadvantages of using amino acid sequence to isolate gene needed

- difficult to find the right mRNA strand because bases are redundant

State advantages of using reverse transcriptase method to isolate needed gene

- will not have introns (so can easily be translated by a bacteria)

State disadvantages of using restriction endonuclease to isolate gene of interest

- restriction - specific recognition sites

- if specific recognition site for endonuclease are within the gene: gene will not be functional

- gene introduced into bacteria cell will have introns which bacteria don't have so translation may lead to not functional protein

Describe characteristics needed in vectors

- be small and self replicating

- not be broken by host cell enzymes

- not stimulate immune response in recipient

- be able to be screened to check gene is inserted

- identifiable by markers to check cells have taken up vector

State examples of vectors used in genetic engeneering

- viruses and plasmids

Why is the same restriction enzyme needed to cut the plasmid as was used to isolate the gene

- so sticky ends are complementary

Describe insertion of DNA into vector

- same restriction enzyme used to cut human gene is used to cut plasmid/vector

- sticky ends made

- gene of interest and plasmid DNA mixed and their complementary sticky ends will base pair with each other

- DNA ligase enzyme joins the sugar-phosphate groups between plasmid and gene/ligasing the 2 pieces of DNA to form a plasmid made of recombinant DNA

- phospho-diester bonds made

Outline how the recombinant DNA is introduced to host cell

- recombinant DNA and bacteria cells are mixed together under correct conditions (medium containing calcium ions)

- some bacteria take up recombinant DNA (only about 1%)

- gene marker such as antibiotic resistance gene is used to identify the bacteria cell that have taken up plasmid

- cloning of recombinant containing bacteria results in multiple copies of recombinant genes

Outline how to increase rate of DNA uptake by bacterial cells

- use calcium chloride salt to increase rate of DNA transformation of heat shocked bacterial cells: Ca2+ ions bind to DNA (-ve charge) and Cl- ions bind to the membrane lipopolysaccharides (+ve charge)

Outline how to identifiy transformed cells

- ensure DNA is inserted within a gene for antibiotic resistance

- add sample of culture on agar plate without antibiotic

- use velvet to have an exact copy of initial colonies

- add to agar plate without antibiotic antibiotic

- disappeared colonies = have taken up desired gene

Outline cloning and production of protein

- industrial fermentors are used for large scale production of clones and the gene product

- conditions are optimum for bacteria to replicate

- plasmids inside bacteria replicate at every cell division, so the gene in the plasmid is also replicated

- as the bacteria grow, their enzymes transcribe the gene within the plasmid and translate the mRNA to synthesis the protein

- product accumulates and is removed from the fermentor for commercial use

State benefits of genetic engineering in bacteria

- large scale production of complex proteins or peptides which cannot be made by other methods

- removal of need to use extracts from mammalian organs (human insulin)

- bacteria have been modified to produce vaccines and treat disease such as fight crohns disease

- env: bacteria have been modified to clean up mercury pollution and detect arsenic in drinking water

Outline concerns over genetic engineering in bacteria

- plasmids are easily transferred and there is the potential for the antibiotic resistance marker genes to be exchanged with other bacteria

- if they are taken into pathogenic species, the infections they cause will be antibiotic resistant

- the possible transfer of oncogenes or gene switches by using fragments of human DNA, that activate proto-oncogenes in recipient cells

- a microorganism with a new gene may become a threat if released into env

- newly introduced gene may disrupt function of other genes in ways not yet understood

State uses of genetic engeneering

- to transfer genes into bacteria

- to transfer genes into plants and animals

- to transfer genes into humans

State common ways plants are genetically modified

- disease and insect resistant crops

- hardier fruits (last longer)

Describe ways genes can be introduced into plants cells

- same steps to make recombinant DNA and insert into vector first

- gene gun: fires gold particles coated with the gene (goes through plasma membrane)

- agrobacterium timefaciens: a bacterial vector containing gene in plasmid

- electroporation: an electric field to increase membrane permeability to allow uptake of the gene

- microinjection: ultra fine needle to inject gene directly into cytoplasm/nucleus (more common with animal cells)

Name bacteria often used to introduce genes into plants cells

- Agrobacterium tumefaciens

Describe agrobacterium timefaciens function without recombinant gene

- soil bacteria

- enters plant through wounds in roots or stem and stimulates tissues to grow in a disorganised way, producing swollen galls

- crown gall disease

What does agrobacterium timefaciens with recombinant DNA do

- cells contain plasmid with tumour inducing gene which is inserted into plant cell chromosomal DNA causing gall formation

Outline how to transform plants with A. Tumefaciens

- plasmid removed from bacteria and T-DNA cut out by a restriction enzyme cutting out tumour forming gene

- foreign DNA cut with same enzyme

- foreign DNA inserted into T-DNA of plasmid

- plasmid reinserted into a bacteria

- bacteria is used to insert T-DNA carrying the foreign gene into the chromosome of plant cell

- plant is generated from a cell clone. All of its cells carry the foreign gene and may express it as a new trait - grown in culture and can keep cloning

State examples of GM crops

- tomatoes and soya

Describe GM soya

- herbicide resistant soyabean

- can be sprayed onto the crop without affecting it but it kills all the weeds

- weed killer breaks down into soil into harmless components

- could work in similar way to antibiotic resistant genes

Describe GM tomatoes

- tomatoes ripen naturally as they produce an enzyme that breaks down pectin

- problems are created due to transport of tomatoes for long distances

- genetically modified tomatoes we developed to suppress production of an enzyme which causes the fruit to soften as it ripens (with gene that blocks expression of another gene)

- this improves keeping quality, increases shelf life

state benefits of genetically modified crops

- superior keeping qualities (longer shelf life), improved flavour

- higher yield - solving food shortages/enabling crops to be grown in drought areas

- a substantial reduction in pesticide/weed-killer use on crops engineered for resistance to fungal pathogens and insect attack - less bioaccumulation, doesn't kill useful organisms

- nitrogen fixing genes into crops such as rice and wheat - increasing nitrates

state concerns around GM crops

- environment : gene transfer to wild species (such as by pollen) + biodiversity

- organic farming pollen from GM crops can find into way into organic fields

- safety: marker genes, antibiotic resistant gene, when consumer eats it might be transferred to bacteria in intestine through conjugation

Why was it important to find and enzyme that would cut the plasmid at only one site?

- cutting at only 1 site is important for controlling the variables that will be reproduced. If enzyme cut at more sites, plasmid might recombine with different DNA fragments

Why was it important to discard any enzymes that cut the plasmid at the replication site

It would not reproduce and transfer genetic information to its host bacteria cell

Why might it be important to cut the DNA strands as closely to the desired gene as possible?

- to make sure that the desired information is transferred to the plasmid without adding unknown or undesirable sequences

What is the human genome project

A collaborative project to sequence the nucleotides in the human genome in order to improve knowledge and understanding of genetic disorders and improve their diagnosis and treatment

How long did human genome project take

- begun in 1990 and ended in 2003 (earlier than expected due to rapid advances in DNA sequencing and computing)

- project is completed but it will take many years to analyse and study data

State aims of human genome project

- identify all genes and which chromosome each is on

- determine the sequence of the 3 billion base pairs in human DNA and store the information in a database

- improve data analysis tools

- transfer related technologies to private sector to develop medical innovation

- address ethical, legal and social issues that may arise

State main findings of human genome project

- humans have 20500 genes, fewer than expected

- more repeated segments of DNA than expected

- fewer than 7% of families of proteins were specific to vertebrates - emphasises the close relationship between all living organisms

How was the human genome sequenced

- Sanger sequencing

- sequenced small sections of DNA at a time and took a year to sequence a million base pairs

What is an alternative method to Sanger sequencing

- Next generation sequencing

Outline next generation sequencing

- mor rapid technique to sequence genome than Sanger sequencing and can sequence an entire genome in a few hours

Which human genomes were sequenced in the human genome project

- randomly chosen anonymous donations given in USA

- second ever human to be sequenced was James Watson

What was the 100K Genome project

- launched to use next generation sequencing to sequence 100K genomes from NHS patients with cancer or a rare disease from members of their families

State aims of 100K genomes project

- create an ethical, transparent programme based on consent

- set up a genomic service for the NHS to benefit patients

- enable medical and scientific discovery

- develop a UK genomics industry

Define genomics

- study of structure, function, evolution and mapping of genomes

State uses of genomics in healthcare

- more accurate diagnosis

- better prediction of effect of drugs and improved drug design

- new and improved treatments for diseases

- may be possible to tailor therapies to individual patients where an individual could have a unique treatment for a common disease based on their genomic data

Uses of genetic screening

- can determine the nature and inheritance of a genetic condition

- can confirm diagnosis

- indicate appropriate treatment

- allow families to make informed decisions about having children with diseases

- identify people at high risk of condition that may be preventable

Concerns about widespread genetic screening

- invasion of privacy

- detection of abnormal alleles in prenatal tests may lead to increase in abortions

- insurance premiums increase or are denied to high risk individuals

State and describe types of genetic screening

- carrier screening: informed choice to have a child

- pre-implantation genetic screening: IVF embryos screened for CF, Huntington's etc

- prenatal diagnostic testing: newborn baby screening

- pre-symptomatic testing: adult onset cancers and Alzheimer's

- confirmation of disease: suspected disease

- forensic/identity testing

Describe cystic fibrosis

- inherited

- lungs and digestive system clogged with thick, sticky mucus

- problems with breathing and digestion from young age

- can eventually stop working

- now identified by screening tests soon after birth

- treatments are available to help reduce problems but average life expectancy is reduced

What can happen as a result of genetic testing to support patients

- genetic counselling can be offered

- parents can decide to not have children if they are both carriers/ have an antenatal test to see of child will be born with disease

- individuals can change lifestyle/attend more regular screening to try reduce chances of certain disorders

- drugs can be designed to target a specific protein

- non-faulty alleles can be used to replace faulty ones - alleles-gene therapy

Ethical concerns with human genome project and genetic screening

- ownership of genetic info and future use

- safeguards required sp genomic info is not used to set/deny insurance premiums, social discrimination, profit

- risk of discrimination and social stigmitization for adult onset disorders may outweigh benefits

- misuse of genetic info

- designer babies (choosing alleles)

State steps of genetic fingerprinting

- extraction

- PCR used to amplify specific fragments containing STRs

- digestion

- seperation

- hybridisation

Describe extraction in genetic fingerorinting

- sample containing DNA mixed with water saturated chloroform or phenol

- protein precipitates out, leaving pure DNA dissolved in water layer

Outline digestion step of genetic fingerprinting

- restriction endonuclease enzymes are added to DNA (produced by bacteria)

- they cut out specific points either side of the STRs (at specific sequence of bases = recognition sequence) so fragments of DNA of different lengths are made

- can digest DNA with more than one restriction endonuclease - generates more fragments

Outline separation stage: gel electrophoresis

- DNA fragments carry negative charge due to phosphate group

- placed at one end of agarose gel

- agarose gel is a matrix with pores which separate DNA fragments according to size - smaller fragments travel further

- they run towards the positive end of the gel at different speeds (largest = slowest)

- they separate into bands

Outline separation stage: southern blotting

- DNA isn't visible, probe is added

- separation of DNA fragments

- agarose gel is soaked in alkaline solution: high pH breaks H bonds in protein structure

- the now single stranded fragments are transferred onto a nylon membrane

- to do this absorbent paper is placed over the nylon membrane and the DNA is drawn up onto the membrane by capillary action

Outline hybridisation step

- DNA probes can be added to bind with DNA STRs and make DNA visible

- probes are single stranded pieces of complementary DNA with radioactive markers at either end

- these probes are complementary to STRs

- Nylon membrane must be put in a solution containing the probes so they can attach

How is DNA visible in genetic fingerprints

- X-ray film is put over the membrane containing the DNA probes

- where probes have bound to STR, radiation will be emitted and fog the film

Uses of genetic fingerprinting

- may be used in human paternity testing

- forensic testing

- can be used for evolutionary studies to determine which species are closely related to one another and common ancestor

State pros for DNA profiling

- technique can be used on samples too small for blood testing

- no invasive method to acquire a sample

- it has reversed wrongful convictions and exonerated falsely accused

Cons of DNA profiling

- could produce wrongful convictions if it is used to influence judges and juries, errors may have occurred in the process

- people conducting tests are not trustworthy

- DNA evidence could be planted at crime scene

- some consider it as a violation of an individual's right to privacy

- DNA profiles held on databases vulnerable to misuse and hacking

What does PCR stand for

- polymerase chain reaction

What percentage of human DNA is made up of exons

- less than 2%

What are introns made up of

- regions of non-coding DNA

- contain repeating nucleotides = short tandem repeats

- length of repeating nucleotides varies

What causes individuals to have different genetic fingerprints

- number of times that the blocks of short tandem repeats are repeated is different

What is a short tandem repeats

- bases of AGCT repeat 6-15 times depending on the allele

- the more times it repeats, the larger the fragment will be

- makes up introns

What is PCR

- polymerase chain reaction technique used to amplify small sections of DNA rapidly

- amplifies short tandem repeats by using a primer (single stranded DNA typically 6-25bp in length) which is complementary to the start of the sequence

State steps of PCR

- strand separation

- primer binding

- strand synthesis

- repeat

Outline strand separation step of PCR

- DNA heated to 95 degrees

- this causes strands to separate (breaks H bonds between base pairs)

Outline primer binding

- sample is cooled to 50-60 degrees and the primers are added in excess to DNA

- they are single stranded pieces of complementary DNA

- they bind to DNA strand

- at 55 degrees, the primers will begin to attach (base pairing) with complementary DNA sequence

Outline strand synthesis

- heated to 70 degrees: enzyme DNA polymerase (heat stable form called Tag) can work at its optimum 70

- DNA polymerase adds complementary nucleotides by forming phosphodiester bonds in sugar - phosphate backbone

State limitations of PCR

- contamination

- error rate

- DNA fragment size

- sensitivity to inhibitors

- limits on amplification

Outline contamination of PCR

- any DNA entering system by accident can be amplified

Explain error rate of PCR

- wrong nucleotide could be inserted

- it makes an error in every 9000 nucleotides

- after 30 cycles this rate becomes 1/300 nucleuotides

Explain limits on amplification

- after about 20 cycles the rate slows down, increase becomes linear (was exponential at the start) and then plateaus

how to distinguish which host cells have taken up empty plasmid for insulin

• plasmid should have antibiotic resitance gene so ones with vector will resist antibiotics

• blue white screening: bacteria grown on medium containing lactose analogue X-gal - white: plasmid with gene, blue: empty