6.3 - manipulating genomes

DNA profiling

a technique used to identify unique DNA patterns in individuals

• also known as genetic fingerprinting

what does DNA profiling rely on?

the fact that, with the exception of identical twins, every person's DNA sequence is distinct. This is due to variation in the sequence and length of unique non-coding, repetitive DNA segments (VNTRs)

VNTRs

• variable number tandem repeats

• short sequences of DNA that repeat many times in a row, but the number of repeats varies between individuals

features of VNTRs

• They are present across the genomes of most eukaryotes.

• They are not involved in protein coding.

• Their length and location are heritable.

STRs

repeated sequences of nucleotides that are smaller than VNTRs and can also be used in DNA profiling

process of DNA profiling

1. DNA extraction - DNA is extracted from a tissue sample and amplified using PCR.

2. DNA digestion - Restriction enzymes are used to cut the DNA into fragments at points near the VNTR sequences.

3. Fragment separation - Gel electrophoresis separates the fragments by size, and they are denatured to produce single strands.

4. Hybridisation - Specific radioactive or fluorescent probes bind to complementary VNTR sequences.

5. Development - The positions of the probes are revealed, creating a barcode-like pattern of DNA bands unique to each individual.

uses of DNA profiling

• Identifying suspects from crime scene DNA (e.g. in blood, saliva, skin cells,)

• Identifying the risk of genetic disorders and predicting their onset and severity.

• Selecting desirable traits in plants and animals for selective breeding while preventing severe inbreeding.

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

limitations of DNA profiling

• Environmental contamination may compromise results.

• Close genetic relatives could have similar fingerprints.

polymerase chain reaction

a method for amplifying DNA fragments rapidly and efficiently

what does amplifying mean in terms of PCR?

a large number of DNA fragments are being produced

what type of technique is PCR?

an automated technique that does not require living cells to rapidly replicate specific DNA fragments. It is sometimes called 'in vitro cloning'

components for PCR

• DNA fragment - the specific target DNA template sequence that needs to be replicated.

• Primers - short sequences of nucleotides that attach to the start and end of the DNA fragment to be copied.

• DNA polymerase - creates new DNA strands by adding DNA nucleotides.

• Free nucleotides

• Thermocycler - This is a device that precisely heats and cools the PCR mixture to facilitate the reaction.

Taq polymerase

a type of DNA polymerase that can withstand high temperatures without denaturing

• it’s used to ensure it remains active throughout the process

First stage of PCR

denaturation - separation of DNA strands

• occurs at 95 degrees 

• Heating the DNA separates the hydrogen bonds between its two complementary strands.

second step of PCR

Annealing - addition of primers 

• occurs at 55 degrees 

• The primers attach to the specific starting points on each of the separated DNA strands by forming hydrogen bonds.

third step of PCR

elongation - DNA synthesis

• occurs at 72 degrees 

• DNA polymerase adds free nucleotides to the ends of the primers, extending the DNA strand to form a complete copy.

Advantages of PCR

• Rapid speed, which is not possible with in vivo cloning

• Low DNA needs - tiny samples can be amplified to produce a large quantity for analysis

Gel electrophoresis

a technique used to separate molecules such as DNA, RNA, or proteins based on size by using an electric current applied to an agarose gel matrix

How to set up gel electrophoresis

1. Insert a gel tray with solidified agarose gel into a gel tank.

2. Ensure the wells are close to the negative electrode to position the gel correctly.

3. Pour a buffer solution over the gel until it is submerged to maintain a constant, suitable pH throughout the experiment.
   

Running electrophoresis on DNA, RNA, and protein samples

1. Mix the DNA or RNA samples with loading dye to make them visible.

2. Carefully deposit equal volumes of each sample into the wells using a micropipette.

3. Touch the micropipette tip to the buffer, not the bottom of the gel, to prevent damaging the gel.

4. Keep a record of which sample is in each well for later analysis.

why do DNA and RNA molecules carry a negative charge?

because of their phosphate groups

what happens during electrophoresis?

1. A voltage is applied across the gel.

2. Fragments of DNA or RNA move towards the positive electrode (anode).

3. The smaller fragments travel faster and thus separate by size.

4. Continue the process until the dye approaches the end of the gel.

how are results from electrophoresis observed?

1. Switch off the voltage and remove the gel from the tank.

2. Apply a stain to the DNA or RNA to reveal the bands of fragments.

3. Assess the migration distances of the bands to approximate the sizes of the fragments.

DNA sequencing 

the process of determining the exact sequence of nucleotides within a DNA molecule

Sanger sequencing

A process used to determine DNA sequences, which relies on the radioactive labelling/fluorescent tagging of bases and gel electrophoresis

Why are fluorescent tags used instead on DNA sequencing?

for safety and efficiency

Human genome project

an international project to map the entire human genome, which used these advanced sequencing and computing techniques

Outline of Sanger sequencing

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

2. DNA is heated to separate it into single strands (denaturation)

3. Primers bind to the single-stranded DNA (annealing) and DNA polymerase builds new complementary strands by adding nucleotides

4. When a terminator base is added DNA synthesis stops, and each is tagged with a unique fluorescent colour.

5. This produces DNA fragments of all possible lengths.

6. These DNA fragments are separated by length using gel electrophoresis.

7. A laser then detects the fluorescent colours of the terminator bases in each fragment to determine their sequence order.

8. With every potential base marked, computer software analyses the fragments to reconstruct the original DNA sequence.

Bioinformatics

involves developing software, computing tools, and mathematical models to collect, store, and analyse biological datasets like the nucleotide sequences of genes and genomes, as well as amino acid sequences of proteins

Computational biology

uses bioinformatics tools and biological data to model biological systems and processes

genomics

applies DNA sequencing and computational biology to study the genomes of organisms

Studying human health and disease through genome analysis

• Sequencing thousands of human genomes has made it possible to identify patterns in our DNA and disease risks.

• Bioinformatics databases offer health professionals information about mutations that may cause genetic disorders.

Sequencing pathogen genomes provides multiple benefits

• Identifying the sources and transmission routes of diseases.

• Detecting antibiotic-resistant strains.

• Developing new treatments and vaccines by identifying potential drug targets.

• Monitoring disease outbreaks.

DNA barcoding

involves comparing the DNA sequence of an unidentified organism to a database of standard ‘barcode’ sequences for known species

advantages of DNA barcoding

• Fast and affordable sequencing.

• The classification of new species.

• Updating of classifications.

• Estimating evolutionary divergence times based on predictable DNA mutation rates to construct evolutionary trees.

genomics

the study of genomes, using DNA sequencing and computational biology to analyse data like base pairs in DNA and protein structures

Proteomics

examines the complete set of proteins produced by the genome (the proteome), including their structure and function

Synthetic biology

involves the design and construction of new biological parts, pathways, and organisms, or the re-engineering of existing natural systems

applications of synthetic biology

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

• Utilising microorganisms and biological systems to produce drugs in an efficient and cost-effective manner.

• Constructing fully artificial genomes.

in vivo cloning

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

first step of forming recombinant DNA

1. A vector is cut open at a specific site using a restriction enzyme, creating sticky ends.

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

3. DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the two strands of DNA, joining the sticky ends of the vector and DNA fragment together.

4. The newly formed combined DNA molecule is known as recombinant DNA.

what does transformation involve?

introducing vectors with recombinant DNA into host cells, transforming these cells. The vectors are usually either plasmids or bacteriophages.

second step of forming recombinant DNA

Transformation involves introducing vectors with recombinant DNA into host cells, transforming these cells. The vectors are usually either plasmids or bacteriophages.

plasmid vectors: Host cells are treated to enhance the uptake of plasmids that have recombinant DNA. Electroporation uses an electrical current to make bacterial membranes more porous, helping plasmids enter bacterial cells

bacteriophage vectors: viruses that infect bacteria. Bacteriophages inject their DNA into host bacterial cells during infection.The phage DNA, now carrying the recombinant DNA, inserts into the host's DNA.

What do marker genes indicate?

which host cells took up recombinant DNA:

1. They are inserted into vectors alongside target genes.

2. Transformed cells are cultivated on selective agar plates.

3. Only transformed cells display the characteristics encoded by marker genes.

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

Marker genes

Different marker genes can identify which host cells took up recombinant DNA

examples of marker genes

• A marker gene for a specific trait, like antibiotic resistance, ensures that only transformed cells form colonies.

• A marker gene that is visible under UV light like green fluorescent protein (GFP).