StemUp: OCR A A level Biology 6.1.3 Manipulating genomes

What are the key components added to the reaction mixture in the chain-termination method of DNA sequencing? (5)

- Single-stranded DNA template (the DNA to be sequenced)

- All four standard nucleotides (A, T, C, G)

- DNA polymerase (to join nucleotides together)

- Primers required for replication

- Fluorescently-labelled modified nucleotides (added to stop replication at certain points)

What happens when a modified nucleotide is added during DNA sequencing? (2)

- Replication is terminated when a modified nucleotide is incorporated into the growing DNA strand.

- This creates DNA fragments of different lengths

How are the DNA fragments separated in the chain-termination method?

- High-resolution electrophoresis is used to separate fragments by size

- Negatively charged DNA moves toward the positive electrode

- Smaller fragments move faster

- Fragments are visualised under UV light, and the base sequence is read from the bottom of the gel upwards

Why does the genome have to be fragmented before sequencing? (3)

- The genome is very large.

- Fewer errors occur with smaller fragments

- It allows the sequencing job to be divided across different times and labs

What are the steps for sequencing an entire genome using the chain-termination method? (6)

1. Genome is cut into smaller fragments using restriction enzymes

2. Fragments are inserted into bacterial artificial chromosomes (BACs)

3. Each BAC is inserted into bacteria

4. Bacteria divide, creating colonies with specific DNA fragments

5. DNA is extracted, cut, and sequenced using the chain-termination method

6. Sequences are put in order to generate the sequence of the entire genome

What is next-generation sequencing and how does it differ from the chain-termination method? (2)

- Next-generation sequencing (high-throughput sequencing) is much faster and automated compared to the original chain-termination method

- It allows entire genomes to be sequenced more rapidly, such as through high-throughput pyrosequencing

How has gene sequencing allowed for genome-wide comparisons between individuals and species? (5)

- Computational biology and bioinformatics are used for genome comparisons

- It helps predict phenotypes from genotypes

- Computerised comparisons can detect mutations linked to diseases

- Genome comparisons help determine evolutionary relationships between species

- Closely-related species share more DNA and have diverged more recently

How has gene sequencing allowed for the prediction of amino acid sequences in polypeptides? (3)

- Sequencing a gene allows prediction of the sequence of amino acids that the gene codes for

- This helps predict the primary structure of a polypeptide

- It enables synthetic biology, allowing the creation of biological molecules from scratch

How has gene sequencing contributed to the development of synthetic biology? (3)

- Involves building biological systems from artificially-made molecules to test their functions

- Helps redesign biological systems for better performance

- Synthetic biology enables the design of new biological systems that don't exist naturally, like creating new drugs

What role do non-coding sequences play in DNA profiling? (4)

- Some parts of the genome contain repeated non-coding base sequences

- The number of repeats at specific loci differs between individuals

- DNA profiling analyzes these repeats using electrophoresis

- The probability of two individuals having the same DNA profile is very low due to the unique number of repeats

What is the first step in carrying out DNA profiling on a sample? (1)

DNA is isolated from the sample

How are multiple copies of DNA made in DNA profiling? (2)

- PCR

- Used to make many copies of the DNA region containing sequence repeats

What is the role of restriction endonucleases in DNA profiling? (1)

Cut the DNA near the repeated regions

How are the DNA fragments separated in DNA profiling? (1)

By electrophoresis

What happens after electrophoresis in DNA profiling? (2)

- Gel is immersed in alkali

- To separate double strands into single strands

What process transfers DNA bands onto a membrane? (2)

- Southern blotting

- Which transfers DNA bands onto a membrane

What is hybridisation in DNA profiling? (1)

When radioactive or fluorescent probes bind to the repeated sequences

What happens to excess probe in DNA profiling? (1)

Excess probe is washed off

How are the DNA fragment positions revealed in DNA profiling? (2)

- X-ray film or UV light detects the probes

- Revealing unique DNA fragment positions

What is a DNA probe? (4)

- A short single-stranded section of DNA (~20 bases long)

- Binds to a complementary DNA sequence

- Radioactively or fluorescently labelled to aid detection

- Probes are 20 nucleotides long to ensure specificity

What are the uses of genetic fingerprinting? (4)

- Forensic science: links a person to a crime scene by comparing DNA profiles.

- Preventing inbreeding: identifies relatedness in plants/animals to avoid mating closely related individuals

- Diagnosing genetic disorders: determines risk or presence of disorders by comparing DNA profiles.

- Preimplantation genetic haplotyping: screens embryos for genetic disorders before implantation in IVF

What is PCR? (3)

- PCR is an in vitro gene cloning technique

- Copies DNA outside of a living organism

- Can produce billions of copies in a few hours

What is PCR used in? (3)

- Forensic science

- Evolutionary biology

- Diagnostics

What is an advantage of PCR? (1)

Works with very small samples

What is a disadvantage of PCR? (1)

Increased chance of contamination

How are DNA fragments amplified using in-vitro cloning (PCR)?

1. Reaction mix: DNA sample, free nucleotides, primers, DNA polymerase

2. Heat to 95°C: Hydrogen bonds break, DNA strands separate

3. Cool to 50-65°C: Primers anneal to DNA strands

4. Heat to 72°C: DNA polymerase adds complementary nucleotides

5. Repeat cycle: DNA doubles each cycle (exponential amplification)

Why are primers needed in PCR? (2)

- Allow DNA polymerase to attach to the DNA strands

- Two different primers are required for the different sequences at the start and end of the target DNA

What happens in each PCR cycle? (2)

- After each cycle, the number of DNA fragments doubles

- E.g., after 1st cycle: 4 fragments, after 2nd cycle: 8 fragments

Why does the amount of DNA produced level off in PCR? (2)

- Nucleotides and primers get used up

- Without these reagents, no more complementary strands can be synthesised

What are the key differences between PCR and transcription? (4)

- Enzyme used: Transcription uses RNA polymerase, PCR uses DNA polymerase

- Nucleotides: Transcription uses RNA nucleotides (uracil), PCR uses DNA nucleotides

- Template: Transcription uses one template strand, PCR uses both strands

- Initiation: Transcription uses start/stop codons, PCR uses primers

What are the differences between PCR and semi-conservative replication? (3)

- Length of DNA: PCR replicates short fragments; semi-conservative replication replicates entire DNA

- Strand separation: PCR uses 95°C heat to separate strands; semi-conservative replication uses DNA helicase

- Primers: PCR requires primers, semi-conservative replication does not

What does electrophoresis separate? (2)

- Separates DNA, RNA fragments, or proteins

- Based on size

What is the first step in electrophoresis of DNA fragments? (2)

- Prepare the gel by pouring agarose gel into a tray

- Creating wells at one end

How do you set up the gel for electrophoresis? (1)

Place the gel in a box or tank with the wells near the negative electrode

What is the purpose of adding a buffer solution in electrophoresis? (1)

Add buffer solution to cover the gel to maintain the pH and conduct electricity

Why is loading dye added to DNA samples during electrophoresis? (2)

- For visibility

- To help the samples sink into the wells

How are DNA samples added to the gel during electrophoresis? (1)

Use a micropipette to add DNA samples to the wells in the agarose gel

How is a current applied to the gel in electrophoresis? (2)

- Connect the box to the power supply

- To run an electrical current through the gel

How does DNA move in electrophoresis? (2)

- DNA, being negatively charged

- Moves towards the positive electrode under the influence of the electric current

How do DNA fragments separate during electrophoresis? (2)

- Smaller DNA fragments move faster through the gel

- Separating based on size

How are DNA bands visualised after electrophoresis? (1)

Using a staining solution that binds to the DNA

What is electrophoresis of proteins used for? (1)

Identifying proteins in urine or blood samples for disease diagnosis

What is a transformed organism? (1)

An organism that has had its DNA altered by genetic engineering

What is recombinant DNA? (1)

DNA containing sections from two species

What is a transgenic organism? (1)

An organism genetically engineered to include a gene from a different species

Why can organisms produce proteins from DNA of another species? (3)

- The genetic code is universal

- Transcription and translation mechanisms are similar across species

- The recipient organism can use the transferred DNA to produce proteins

How is the desired DNA fragment isolated during genetic engineering? (2)

- The DNA fragment containing the desired gene

- is isolated using restriction endonucleases

What are restriction endonucleases and their function? (2)

- Enzymes that cut DNA at specific sequences of bases

- Recognition site

What is a palindromic sequence in DNA? (2)

- A nucleotide sequence that reads the same from 5'-3' direction on one strand

- As it does 3'-5' on the complementary strand

What are sticky ends in DNA? (1)

Overhanging sequences of nucleotides exposed after a restriction enzyme cuts the DNA in an oblique manner

Why do restriction endonucleases cut DNA only at specific places? (2)

- The shape of the recognition site is complementary

- To the enzyme's active site

How can restriction endonucleases be used to produce DNA fragments? (3)

1. DNA is incubated with a specific restriction endonuclease

2. Which cuts the DNA fragment through a hydrolysis reaction

3. Leaving sticky ends that can join to other DNA fragments with the same sticky ends

How is the DNA fragment containing the desired gene inserted into a vector? (1)

Using restriction endonucleases and DNA ligase (in vivo cloning)

What is a vector in genetic engineering? (2)

- Something used to transfer DNA into a cell

- The most common vectors are plasmids and bacteriophages

How is a DNA fragment inserted into a plasmid? (4)

1. The plasmid DNA is cut open with the same restriction endonuclease that isolated the DNA fragment

2. Leaving complementary sticky ends

3. The DNA fragment is mixed with the plasmid DNA and DNA ligase

4. Which joins the sticky ends to form recombinant DNA

What is the end result of inserting a DNA fragment into a plasmid? (2)

- A recombinant plasmid carrying the gene of interest

- Consisting of vector DNA and the DNA fragment

How is the gene transferred into bacteria using a vector? (1)

The vector with recombinant DNA is used to transfer the gene into bacterial cells

What is electroporation and how is it used to introduce recombinant plasmids into bacterial cells? (2)

- Technique where bacterial cells and plasmid vectors are mixed and placed in an electroporator

- Which creates an electrical field that increases the cell membrane's permeability, allowing plasmids to enter

What are the advantages of PCR over in vivo gene cloning? (5)

- PCR is safer (uses DNA/enzymes, not whole cells)

- Quicker: takes hours, whereas in vivo cloning takes weeks

- Less equipment: requires only a tube and heat block

- Less labour-intensive

- Can use low-quality DNA (e.g. forensic DNA)

What are the disadvantages of PCR over in vivo gene cloning? (4)

- In vivo is less prone to mutation (Taq polymerase in PCR may introduce errors)

- In vivo is cheaper (bacterial growth materials vs. expensive PCR chemicals)

- In vivo can clone longer DNA pieces, while PCR is limited in size

- In vivo cloning is less technically complex (less critical conditions)

What are the positive and negative ethical issues associated with genetically modifying plants? (3)

- Positive: Reduces pesticide use, which is better for the environment and cheaper

- Negative: Encourages monoculture, decreases biodiversity, and increases susceptibility to disease.

- Genetically modified genes may spread to wild populations

What are the positive and negative ethical issues associated with genetically modifying animals? (2)

- Positive: Can produce large quantities of drugs, making them more accessible

- Negative: Potential harm to animals, reduces animals to commodities, and concerns about animal welfare

What are the positive and negative ethical issues associated with genetically modifying pathogens? (2)

- Positive: Large-scale production of human forms of hormones/antibiotics. Research can treat previously untreatable diseases

- Negative: Risk of infection for scientists, reversion of modified pathogens causing outbreaks, and potential use in biological warfare

What are the positives of genetically engineered organisms being owned by big companies? (2)

- Owners of the patent receive money from selling the product

- Encouraging faster development of genetically engineered products

What are the negatives of genetically engineered organisms being owned by big companies? (1)

Farmers in poor countries may not be able to afford the patent for genetically modified seeds.

What is gene therapy and what does it aim to treat? (2)

- The process of altering defective genes inside cells to treat genetic disorders and cancer

- Defective genes are usually mutated alleles

How can gene therapy treat diseases caused by two mutated recessive alleles? (1)

By adding a working dominant allele

How can gene therapy treat diseases caused by one mutated dominant allele? (2)

- Silence the dominant allele by inserting a piece of DNA into the middle of the allele

- Preventing it from producing a functional protein

What vectors can be used to insert new DNA in gene therapy? (1)

Vectors such as altered viruses, plasmids, or liposomes can be used to insert new DNA

What is somatic gene therapy? (2)

- Somatic therapy involves altering alleles in body cells

- It doesn't affect sex cells, so offspring could still inherit the disease

What are the limitations of somatic therapy? (3)

- Effects may be short-lived, requiring multiple treatments.

- It can be difficult to target specific cells

- The procedure may be invasive or dangerous

What is germ line gene therapy, and why is it controversial? (3)

- Alters alleles in sex cells,

- Affecting all future offspring

- It is currently illegal.

What are some risks associated with gene therapy? (3)

- The body could identify vectors as foreign bodies, leading to an immune response

- An allele might be inserted in the wrong place, causing problems

- Could be over-expressed, producing too much of the protein