DNA technology

What is bioinformatics?

The storage and analysis of large datasets relating to biological material (DNA sequences, protein sequences etc) 

Is eukaryotic easy or difficult to sequence?

In eukaryotes, sequencing the genome (all of the genetic material of a cell/organism) does not allow you to easily predict the proteome (all of the proteins that can be produced by the cell/organism). 
This is because: 
a) Much of the DNA is non-coding (98% in humans) 
b) Many of the genes are not being expressed – just because the gene is there doesn’t mean that the protein is there too 

What is genetic engineering?

DNA from one organism is isolated and then placed into/combined with the genome of another organism, also called recombinant DNA technology. Leads to transgenic organisms or genetically modified organisms (GMOs) 

What is the Human Genome Project?

It is an international scientific project which has successfully determined the sequence of bases of a human genome, running from 1990-2003

Key steps in genetic engineering

1. Extraction of DNA e.g. using detergent to break down the phospholipid bilayer of the plasma membrane and nuclear membrane and release the DNA  

2. Isolation of the gene – restriction endonucleases are used to cut out the gene from the human DNA/genome. These enzymes cut at very specific locations 

3. Insertion of gene into a vector – the vector (e.g. a plasmid from E Coli) is cut open using the same restriction enzyme (to have complementary sticky ends and allow hydrogen bonds to form) and then the gene is inserted into the vector using the enzyme DNA ligase. DNA ligase catalyses the formation of a phosphodiester bond between the gene and the vector. This makes recombinant DNA 

4. Transformation – the recombinant DNA is transferred into a host cell (e.g. the E. coli is treated with heat or chemical shock to open up the membrane). In a tissue culture there is the issue that not all cells will become successfully transformed 

5. Identification – check cells have been successfully transformed using gene markers e.g. antibiotic-resistant gene. Cells that have transformed successfully will have both the insulin gene and the antibiotic resistance gene, as they are in the same plasmid. Those that did not successfully transform will not have the insulin gene or the antibiotic resistance gene because they did not take up the plasmid (which contains both genes). When introduced to antibiotic, only the cells that did not transform effectively will be killed (could alternatively use a fluorescent gene rather than antibiotic resistant) 

6. Growth/cloning: the transformed cells are cloned. Secreted insulin is collected and purified (issues of binary fission is that not all daughter cells may have the plasmids – however rapid cell division decreases this frequency) 

Restriction endonucleases

• Found naturally in bacteria, helps bacteria to protect themselves against foreign viral DNA (bacteriophages) 

• Cut DNA at very specific base sequences that are palindromic

• Breaks/hydrolyses phosphodiester bonds

• e.g. EcoRI cuts ONLY at GAATTC, BamHI cuts only at GGATCC, which is palindromic  

• Most restriction enzymes make a staggered cut in the two chains, forming sticky ends. Sticky ends are only complementary to ends that have been cut by the same restriction enzyme. However, some restriction enzymes cut straight across both chains, forming blunt ends 

Issues with the process of genetic engineering

• Genes of bacteria don’t have introns, therefore do not have sufficient enzymes to splice introns out of human genes that are inserted into plasmids. The introns will not code correctly for the right protein, as it cannot recognise introns.  
  Mature insulin mRNA is extracted from cells (meaning only exons and no introns) and copied to make single-stranded complementary cDNA using the enzyme reverse transcriptase. DNA polymerase is then used to make a double stranded version of the cDNA. DNA produced from the mRNA is put into the bacteria, as it then has no introns 
  - Reverse transcriptase makes single stranded cDNA from mature mRNA 
  - DNA polymerase synthesises second DNA strand to the cDNA 
  Disadvantages of this method as that mRNA can only be taken from a cell that is actively making insulin, the pancreatic cells. Single stranded mRNA is also vulnerable to degradation, so it is difficult to work with. 
  Alternatively, you can chemically synthesise the gene you want (including missing out the introns) 

• Prokaryotic cells don’t have membrane-bound organelles, for example a Golgi body, which is important for protein secretion 

How to chemically synthesise a gene

The ‘gene machine’

• Work backwards from the desired protein primary structure 

• Input desired base sequence for amino acids into a computer  

• Can’t build a whole gene in one go, so instead short overlapping sections of nucleotides (oligonucleotides) are chemically built 

• These sections are then joined together by DNA ligase

• Thousands of copies are made by amplification using PCR

• These can then be inserted into the plasmid (by adding restriction enzyme sites on the ends of the gene) 

Origin of replication

where plasmids start DNA replication. Ensures the plasmid replicates quickly to increase the likelihood of daughter cells inheriting the plasmid 

Selectable marker

fluorescent gene used to reduce risk of false positives  

Promoter

the promoter region must be ‘upstream’ of the gene. It allows RNA polymerase and transcription factors in the host cell can bind and transcribe the gene into mRNA

What are three reasons why a host cell might not take up a recombinant plasmid?

1. The recombinant plasmid doesn’t get inside the cell 

2. The plasmid re-joins before the DNA fragment entered  

3. The DNA fragment sticks to itself, rather than inserting into the plasmid 

What are two ways in which you can identify if a cell has transformed correctly and successfully uptaken the recombinant plasmid?

Use of marker genes on the plasmid

• antibiotic resistance genes (replica plating)

• genes coding for fluorescent proteins (GFP) or coding for enzymes

Overview of replica plating as a method of identification

• plasmid will include two different antibiotic resistance genes e.g. ampicillin and tetracycline

• the gene of interest is inserted inside the resistance to tetracycline gene and disrupts it. Therefore this gene will no longer create a functional protein (although will still be resistant to ampicillin) 

• bacteria that are able to grow on agar with ampicillin have uptaken a plasmid, as they must contain an ampicillin-resistance gene (however the plasmid may not necessarily be recombinant and have the gene of interest)

• transfer some of the bacteria to agar with tetracycline. Any colonies still growing must contain bacteria without the gene of interest, as there is no DNA fragment to disrupt the tetracycline resistance gene. Bacteria resistant to ampicillin but not tetracycline contain the recombinant plasmid  

How do fluorescent markers work as a method of identification?

• Jellyfish contain a gene which codes to create a green fluorescent protein. Plasmid created with GFP gene in  

• DNA fragment (insulin gene) is inserted into the middle of the GFP gene. This disrupts it and prevents GFP production 

• Grow the bacteria on agar. When exposed to UV light, the ones with GFP gene intact glow. Therefore, only non-glowing colonies contain the recombinant plasmid 

What is the Polymerase Chain Reaction

• PCR is a commonly used technique in molecular biology to make millions of copies of a stretch of DNA of interest  

• In vitro DNA replication  

• It amplifies ‘target DNA’ (without amplifying DNA that you’re not so interested in)

• It uses DNA polymerase  

• Takes place inside a machine called a thermocycler 

Components of PCR

• DNA sample containing the target DNA 

• Taq DNA polymerase: DNA polymerase that has been made by bacteria rather than humans (from bacterium Thermus aquaticus, which is found in hot springs and therefore has an optimum temperature of 72 degrees. Ensures the DNA polymerase doesn’t denature) 

• DNA primers: artificial complementary sequences of DNA that latch onto the ends of the desired sequence so that DNA polymerase can bind   

• Free nucleotides (an excess) 

• (pH buffer)

• (Cofactors  

Describe how restriction endonuclease and DNA ligase are used to insert a gene into a plasmid [2]

1. Restriction endonuclease cuts plasmid
   (or)
   Restriction endonuclease produces sticky ends

2. DNA ligase joins DNA/gene and plasmid
   (or)
   DNA ligase joins sticky ends

Why does DNA replication slow down eventually when using PCR?

• Runs out of free nucleotides

• May run out of DNA primers

Temperatures of PCR

1. 94 = Denaturation. This stage breaks the hydrogen bonds holding the DNA strands together, causing the two strands to separate (need more heat if there are more C-G base pairs, as they have 3 hydrogen bonds) 

2. 54 = Annealing of primers. The DNA primers attach to complementary bases at either end of the DNA 

3. 72 = Extension. Taq DNA polymerase attaches the free nucleotides by phosphodiester bond to synthesise a complementary strand of DNA. This is the optimum temperature for taq DNA polymerase 

After one round of PCR you will have two copies of the DNA fragment

Advantages of PCR

• Rapid – 100 billion copies of DNA can be made within hours  

• Doesn't require living cells therefore is quicker and less complex techniques are needed (don’t have to grow cells or look after cells – reduces effort and costs) 

• PCR has no ethical issues

• Only need a tiny amount of starting DNA (can have one single cell’s worth of DNA) 

Cons of PCR

• Taq polymerase is quick, so it can make mistakes in copying and introduce mutations – taq polymerase does not proofread well. Bacteria in in-vivo cloning don’t make mutations nearly as much 

• It only amplifies DNA – can't create transgenic organisms. It can be useful for finding out more about DNA but cannot be used to directly produce proteins or drugs for commercial or medical use 

• It’s quite easy to amplify DNA beyond your target DNA, where DNA polymerase continues beyond the gene of interest – so the DNA can become contaminated with unwanted DNA. Using restriction enzymes at the end of primers and plasmids reduces the risk of contamination and makes sure only your specific gene of interest is cut out 

Pros and cons of in-vivo cloning

Pros

• creation of recombinant DNA rather than just making exact copies

• Lower risk of mutations when using bacteria (compared to PCR)

Cons

• Time consuming

• Living cells so requires monitoring of cell growth

Benefits of using stem cells

• (Embryonic) Due to ability to differentiate into multiple types of cells, there is a huge potential in treating disease and producing transplant tissues or organs – e.g. in generating insulin producing beta cells for type 1 diabetes. 

• Organs developed from a patient’s own stem cells (or iPS cells) reduce risk of organ rejections 

Risks of using stem cells

• Immune rejection risk of embryonic stem cells from body in transplant if cells are not genetically matched.  

• Stem cells cultured in the lab could become contaminated with viruses and risk transmission to patients 

• Can form tumours if cell division is not controlled properly and stem cells accumulate. 

• There are low numbers of stem cell donors 

Social and ethical issues of stem cells

• Very expensive, not an option for everyone

• Lack of peer-reviewed clinical evidence of the success of stem cell treatments. Must educate the general public on the use of stem cells 

• Embryonic stem cells introduce concerns as it involves the destruction of embryos and whether they have human rights.  

• Sourced from embryos during IVF treatment which raises questions regarding who ‘owns’ the embryo or gives permission for it 

Benefits of using genetically engineered (transgenic) organisms

• GMOs can be engineered to grow faster and become more resistant, increasing crop yields and productivity. Also, positive economic implications 

• GMOs can be nutritionally enhanced to address malnutrition and food production 

• Uses in gene therapy and production of treatments and bioremediation – degrading plastics 

• More precise than selective breeding, inserting specific genes without unwanted characteristics 

Environmental issues of transgenic organisms

• GM crops may reduce biodiversity for future generations, or become ‘superweeds’ and invade natural habitats bordering farmland 

• Development of resistance for the introduced genes in the wild population via gene flow – potential ecological effects to non-targeted species and food webs 

Social/ethical issues of transgenic organisms

• May not be affordable for all farmers or developing countries – biotech companies may charge farmers more money 

• Use of GMOs in the wrong hands – bioweapons, new pathogens 

• Impact upon other economies and global trade across the world, wealthy countries might override trade industry 

• Lack of long-term research on the effects of GMOs in food production, and whether it should be consumed if effects are unknown

• Without appropriate labelling the consumer cannot make an informed decision about the consumption of GM foods 

• Ethical concerns associated with genetically modifying animals  

• Risk of eugenics, social gene engineering – designer babies 

What is DNA profiling/genetic fingerprinting?

• Looking at patterns in the DNA of an individual for identification

• In your non-coding DNA, there are loads of tiny sequences repeated over and over again, known as tandem repeats. They are found in the same locations in everyone, but the number of repeats varies between individuals – called variable number tandem repeats (VNTRs) 

• VNTRS are what scientists look for when drawing up a DNA profile of someone 

What does gel electrophoresis do?

Separates DNA, RNA and protein molecules based on size/mass. DNA carries a negative charge, and small fragments are able to travel more quickly than large fragments towards the positive electrode, the anode (PANiC)

DNA is separated by mass, but separating proteins is more difficult as two proteins of the same mass may have different tertiary structures and different charges – both of these will affect how the protein moves through the gel. Proteins must be heated (denature/break bonds in tertiary structure) and then added to a chemical substance with a negative charge so that the proteins are equally charged  

Process of gel electrophoresis

1. Make gel  

2. Add DNA samples to the wells  

3. Run gel under buffer (including salts and ions so that current can flow) for 30 mins with electric current flowing. Run with a DNA size standard of known DNA fragment sizes to allow comparison  

4. Stain DNA and visualise e.g. under a UV light 

Genetic fingerprinting using STRs requires amplification of the STRs using the PCR. The short base sequences either side of a specific STR are known.
Explain the importance of knowing these base sequences

1. for primers

2. for DNA/Taq polymerase
   Primers stop original DNA strands rejoining

Mutations in genes that code for tumour suppressor proteins can cause cancer
Explain how [3]

1. change in DNA base sequence/triplet

2. change in sequence of amino acids/change in tertiary structure

3. rapid/uncontrollable cell division

Genetic testing, using DNA from saliva, can screen for all known harmful mutations in genes. Describe how this DNA could be screened for all known harmful mutations [4]

1. use of PCR to amplify DNA

2. cut DNA using restriction endonucleases

3. separate DNA fragments using electrophoresis

4. addition of labelled DNA probes and binding (by DNA hybridisation)

5. mutations identified by fluorescence/radioactivity
   (or) compares positions/bands to known DNA sample with harmful mutations

Extended process of genetic fingerprinting

• DNA is extracted from a cell using detergent, and is amplified using PCR 

• Restriction enzymes are added to cut the DNA into different length fragments with sticky ends. The lengths of the VNTR sequences are different in different people, so different sized fragments are produced in different people 

• These fragments are separated by size using gel electrophoresis. DNA fragments are injected into wells and an electric current is applied along the gel. DNA is negatively charged so it is attracted to the positive anode of the gel. The smaller the fragment, the faster it moves 

• The separated DNA fragments on the gel are transferred to a special nylon membrane (DNA will stick to this membrane and be stable) 

• Radioactive or fluorescent DNA probes of known sequence are added to the nylon membrane, which is treated to make the DNA single stranded. The probes have base sequences which are complementary to the VNTRs, allowing them to hybridise (bind). The probe will give off a signal once bound to the DNA

• The nylon sheet is placed under X-ray film or UV light. The radioactive probes on the DNA fragments expose the film. This produces visible patterns of dark and light bands which are unique to each individual.

• The position of fragments is then analysed and compared to other samples. The patterns of the bands are unique to every individual  

• Genetic fingerprinting is used to solve crimes, carry out paternity tests, give medical diagnoses to look for disease markers (e.g. alleles for cancer), measure genetic diversity in a population and for breeding programmes 

What are DNA probes?

• short, single-stranded DNA molecules of known base sequences that are labelled either with a fluorescent tag or a radioactive isotope

• bases are complementary to your region of interest e.g. VNTR sequence. When the probe hybridises to the region of interest, it will give off a signal (must wash off any unbound probes to ensure no false positives)

• artificially made using ‘gene machine’

Why must the nylon membrane be treated?

• The DNA must be single stranded, as hybridisation of DNA probes will only happen if the complementary DNA bases are exposed

• This is usually done by using heat to break the hydrogen bonds

Other than genetic fingerprinting, what else can DNA probes be used for?

• detection of mutant alleles e.g. in disease diagnosis

What are the function of primers?

Marks the region of DNA that needs to be copied/amplified, showing DNA polymerase where to start replication. Primers enable replication to start