6.3- Manipulating the Genome

Sanger Sequencing Method

1. DNA mixed with primers, DNA polymerase, normal nucleotide bases and terminator bases

2. DNA split into single strands and copied multiple times

3. DNA polymerase adds nucleotides to single stranded template

4. when terminator base added to DNA, synthesis stops→ tagged with fluorescent colour

5. produces DNA fragments of all possible lengths

6. Fragments separated by length→ gel electrophoresis

7. laser detects fluorescent colours of bases to determine their sequence order

advancements in sequencing

• parallel sequencing→ allows simultaneous sequencing of millions of DNA fragments

• Exponentially increased speed→ bacterial genome can be sequenced in less than 24 hours

• reduced costs→ can sequence genomes of more organisms

gel electrophoresis

• separates DNA fragments by length

• Phosphate groups negatively charged→ attracted to anode

• shorter fragments move at faster rates→ fragments move different distances over time

what happens during electrophoresis

• voltage applied across the gel

• fragments of of DNA OR RNA move towards anode

• fragments travel at different speeds and therefore separate by size

analysing results from gel electrophoresis

• stain applied to DNA/RNA to reveal bands of fragments

• Migration distances assessed to approximate sizes of fragments

genetic fingerprinting

• also known as DNA profiling

• used to identify DNA patterns in individuals

• helps identify individuals in forensics or family relationships

• relies on the fact that nearly every person’s DNA sequence is distinct

variable number tandem repeats

• unique, non-coding, repetitive DNA segments

• variation in sequence and length of VNTRs= distinct DNA sequences

key features of VNTRs

• present across genomes of most eukaryotes

• not involved in protein coding

• very variable in sequence and length among individuals

• length and location inheritable

• high similarity in VNTR patterns= closely related

creating a genetic fingerprint

1. DNA extracted from tissue and amplified using PCR

2. Restriction enzymes use to cut DNA into fragments at points near VNTR sequences

3. gel electrophoresis separates fragments by size and they are denatured to produce single strands

4. specific radioactive/ fluorescent probes bind to complementary VNTR sequences

5. Positions of probes are revealed→ makes pattern of DNA bands unique to individual

uses of genetic fingerprinting technology

• paternity testing

• identifying suspects from crime scene DNA

• supporting criminal convictions with match probability calculations

• identifying risk of genetic disorders and predicted severity

• selecting desirable traits in selective breeding

• evaluating genetic diversity by comparing variety of genetic fingerprints within a population.

limitations of genetic fingerprinting

• environmental contamination can compromise results

• close genetic relatives could have similar fingerprints

• assumptions about variation underpin prob. calculations→ not always prove guilt or causation

polymerase chain reaction

• a method for amplifying DNA fragments rapidly and efficiently

• does not require living cells to rapidly replicate specific DNA fragments

• also known as in vitro cloning

components required for PCR

• DNA fragment→ target DNA template sequence that needs to be replicated

• Primers→ short sequences of nucleotides that attach to start and end of fragment

• DNA polymerase→ must be able to withstand high temps e.g. Taq polymerase

• Free nucleotides

• Thermocycler→ device that precisely heats and cools PCR mixture to facilitate reaction

stages of PCR

1. Separation (denaturation)→ 95°C:

   • separates hydrogen bonds between two complementary strands

2. Addition of primers (annealing)→ 55°C:

   • H bonds form between primer and starting points on each of the strands

3. DNA synthesis (extension)→ 72°C:

   • DNA polymerase adds free nucleotides to ends of primers, extending DNA to form complete copy

Advantages of PCR

• rapid speed

• Highly precise

• Low DNA needs

• No cells needed

uses of DNA sequencing

• Computational biology, bioinformatics and genomics

• genome analysis

• genome comparison

• synthetic biology

bioinformatics

Involves developing software, computing tools and mathematical models to collect, store and analyse biological datasets e.g. nucleotide sequences of genes and genomes

computational biology

uses bioinformatics and biological data to model systems and processes

genomics

studying genomes of organisms

genome analysis

• can be used to study human health and disease

• can identify patterns in DNA and disease risks

• Pathogen genomes can be sequenced:

  • identifying sources and transmission roots of diseases

  • detecting antibiotic resistant strains

  • developing new treatments and vaccines

  • monitoring disease outbreaks

comparing genomes

• indicates common ancestry using similarities in DNA barcoding:

• Advantages of DNA barcoding:

  • fast and affordable sequencing

  • classification of species

  • estimating evolutionary divergence times

genomics and proteomics

• genomics→ the study of genomes using DNA sequencing

• Proteomics→ examines complete set of proteins produced by genome, including structure and function

• number of proteins can exceed number of genes (e.g. mRNA splicing, post-translational modifications)→ makes relationship between genotype and phenotype complex

synthetic biology

• design and construction of new biological parts, pathways and organisms

• can be used for:

  • synthesising functional genes to replace faulty ones as treatments for genetic disorders

  • utilising microorganisms and biological systems to produce drugs in efficient manner

  • constructing artificial genomes

what is genetic engineering

• the deliberate manipulation of genetic material to modify an organism’s characteristics

• often involves gene transfer

what is recombinant DNA

• DNA that is altered to contain nucleotides from 2 different organisms

• allows DNA fragments to be transferred between organims

• organisms that receive transferred DNA fragments are called genetically modified/ transgenic organisms

stages in gene transfer

1. desired gene is identified and isolated

2. multiple copies of the gene are made using PCR

3. gene inserted into vector

4. vector delivers gene into cells in a different organism

5. cells with new gene identified e.g. by using marker

6. cells with new gene are cloned

methods of producing DNA fragments

1. making complementary DNA (cDNA) using reverse transcriptase and mRNA

2. Cleaving DNA from a donor organism with restriction enzymes

how does reverse transcriptase produce DNA fragments

1. mRNA is extracted from cells

2. mRNA is reverse transcribed using reverse transcriptase enzyme and DNA nucleotides

3. makes a cDNA strand identical to original DNA strand and cDNA is isolated from mRNA strand

4. cDNA, free nucleotides and DNA polymerase can form other strand of DNA, reforming desired gene

using restriction enzymes to cut DNA

• restriction endonuclease used

• can recognise and cut DNA at specific palindromic nucleotide sequences to isolate gene fragments

process of using restriction enzymes

1. DNA incubated with chosen restriction enzyme

2. restriction enzyme identify palindromic sequences in DNA double helix and cut double stranded DNA if their recognition sequence is present- allow enzyme to separate fragment from rest of the strand.

3. Enzymes cut target gene fragment via hydrolysis

4. different restriction enzymes cut at different sequences based on their active site shape

sticky ends

• short overhanging sequences of unpaired bases that can bind to other DNA fragments when they are inserted into vectors

• often found DNA cut by restriction enzymes

steps in forming recombinant DNA

1. inserting DNA fragments into vectors

2. transferring recombinant DNA into host cells

3. identifying transformed host cells

in vivo cloning/ in vivo DNA amplification

process of producing large quantities of a target DNA fragment in living cells

inserting DNA fragments into vectors

1. vector cut open at specific site using restriction enzyme- creates sticky ends

2. same restriction enzyme used to cut target DNA fragment, creating complementary sticky ends

3. DNA ligase forms phosphodiester bonds between sugar and phosphate groups on two strands of DNA- joins sticky ends of vector and DNA together

4. newly formed combined DNA molecule= recombinant DNA

transferring recombinant DNA into host cells

• can be done using:

  • plasmid vectors

  • bacteriophage vectors

plasmid vectors

• small, circular DNA molecules typically found in bacteria

• host cells treated to enhance uptake of plasmids that have recombinant DNA

  • e.g. applying Ca²⁺ and temp. shifts= bacterial membranes more permeable to plasmids

• electroporation uses electric current to make bacterial membranes more porous- helps plasmids enter bacterial cells more easily

bacteriophage vectors

• viruses that infect bacteria

• inject their DNA into host bacterial cells during infection

• phage DNA, now carrying recombinant DNA, inserts into hosts DNA

identifying transformed host cells

• not all host cells may uptake DNA- important to identify which cells have been transformed successfully

• marker genes indicate which host cells took up recombinant DNA

  1. Inserted into vectors alongside target genes

  2. transformed cells cultivated on selective agar plates

  3. only transformed cells display characteristics encoded by marker genes

  4. transformed cells can be cultured to mass produce target DNA fragment through cellular replication

types of marker genes

• for a specific trait e.g. antibiotic resistance, ensures only transformed cells form colonies

• visible under UV light like green fluorescent protein

  • inserting marker gene within green fluorescent protein gene inhibits fluorescence if it is successfully incorporated

• marker gene coding for an enzyme that alters colour of a specific substrate

genetically modified crops

• can make them resistant to herbicides or insects

• e.g. soy beans have been modified to include gene that produces protein that is toxic to many insect pests but is harmless to humans

advantages of GM crops (5)

• less labour than traditional herbicides and pesticides

• cheaper than traditional herbicides and pesticides

• herbicide resistant GM crops allow use of herbicides to kill weeds without harming the crop, increasing yield

• insect resistant GM crops less likely to be killed by pests

• insect resistant Gm crops reduce need for pesticides- better for environment

disadvantages of GM crops (5)

• encourage monocultures= less biodiversity

• herbicide resistant GM plants may crossbreed with wild plants- makes herbicide resistant weeds

• loss of traditional crop varieties reduces genetic diversity- makes crops vulnerable to disease/ climate changes

• non-target insects could be harmed by toxin coded for by insect resistant GM crops

• insect pests may evolve resistance to toxins

advantages of GM animals

• improves quality e.g. enhancing disease resistance

• improves quantity e.g. enable year-round reproduction

• improves productivity e.g. faster growth

pharming

• production of pharmaceuticals and human medicines by inserting human genes into other animals

• allows for pharmaceutical proteins to be extracted from GM animals’ milk or blood at high yields

advantages of pharming

• enables production of rare treatments

• makes drugs more accessible

disadvantages of pharming

• animal welfare concerns

• can lead to animals being viewed solely as commodities

creating GM microorganisms

• can be used in development of new treatments for diseases e.g. poliovirus can be engineered to target cancer

advantages of GM pathogens

• offers potential treatments for diseases previously deemed incurable

• can produce essential medicines e.g. insulin

• useful in creating vaccines

• facilitates creation of DNA libraries for research

disadvantages of GM pathogens

• carries risk of accidental infections and disease outbreaks

• danger that engineered pathogens could revert to original harmful form

• could be misused in biological warfare

ethical concerns with GMOs

• legal patenting of engineering engineered organisms raises questions about global access

• especially affects smallholder farmers in developing countries who may be unable to afford patented seeds due high costs and legal barriers

how does gene therapy treat genetic disorders

1. identify abnormal gene responsible for disorder

2. engineer normal, functional version of this gene by removing it from health cells or synthesising it in lab

3. deliver normal allele to nuclei of target cells using vector

4. ensure gene is successfully integrated into cells’ DNA and expressed correctly

main approaches to using gene therapy

• counteracting recessive disorders:

  • add functional dominant alleles

  • silences non-functional recessive alleles

• silencing faulty dominant alleles

  • insert DNA sequences that inactivate harmful dominant alleles

  • prevents dominant allele functioning properly and causing harm

somatic gene therapy

• replaces mutant alleles with healthy alleles in affected somatic (body) cells to treat diseases

• alters somatic cells e.g. lungs in cystic fibrosis

• impacts specific tissues and organs only

• genetic modifications not inherited by offspring

germline gene therapy

• involves inserting healthy allele into germ cells or embryos to prevent genetic diseases from birth

• alters egg and sperm cells or embryos

• influences all cells within body when inherited

• modifications be passed down to future generations

ethical benefits of gene therapy

• extends lives by treating diseases

• enhances quality of life

• germline GT= carrier parent can have children free from genetic disorders

• reduces overall disease burden in population

ethical issues of gene therapy

• potential misuse for enhancing aesthetic attributes rather than medical need

• risk of causing unintended harm

• diverts scarce healthcare resources

• high cost- restricted access?

issues with somatic gene therapy

• delivering health alleles to cells is challenging

• getting healthy alleles into nucleus is challenging

• maintaining the expression of healthy alleles is challenging

• effects are short term as somatic cells have a limited lifespan and are replaced by cells with faulty allele

issues germline gene therapy

• the rights of unborn child are violated as they cannot provide consent

• it causes irreversible changes, long term outcome of which aren’t fully understood

• it could used for non- therapeutic enhancements such as selecting desirable traits