Lecture 6 - Manipulation of DNA, Use In Therapy and Diagnosis:

22-24 - Describe the process of DNA sequencing (6) (Diagram)

1. DNA is normally replicated using deoxynucleotides (dNTPs) 2. DNA polymerase incorporates nucleotides by formation of phosphodiester bond between 5' PO4 group of nucleotide being incorporated and 3' OH on previous nucleotide 3. DNA is normally replicated using deoxynucleotides (dNTPs) 4. DNA polymerase incorporates nucleotides by formation of phosphodiester bond between 5' PO4 group of nucleotide being incorporated and 3' OH on previous nucleotide 5. DNA sequencing uses dideoxynucleotides (ddNTPs) which do not have a -OH on the 3' carbon 6. DNA polymerase cannot incorporate any further nucleotides after incorporation of a ddNTP as no 3' OH to form phosphodiester bond

35-43 - Summarise the 3 main stages of the polymerase chain reaction (3)

1. Denaturation: the reaction is heated to ~ 95°C to denature the DNA into single strands 2. Primer annealing: the reaction temperature is reduced to ~ 45-68°C to allow primers to hybridize to their complementary sequences in the target DNA 3. Primer extension: the reactions temperature is raised to 72°C to allow Taq polymerase to synthesize DNA

12 - Describe the process in which DNA is cloned (5)

1. To clone a DNA fragment the fragment and plasmid are cut with the same restriction enzyme. 2. Both plasmid and fragment will have same complementary cohesive ends 3. Cut DNA fragments and cut plasmid are mixed together 4. DNA fragments will anneal to cohesive ends in plasmid DNA ligase will ligate the DNA fragment and plasmid together to form recombinant DNA molecule.

50-52 - Describe the process of the DNA Microarray (6) (Diagram)

1. ssDNA (oligonucleotides) matching all genes in genome are spotted onto microarray slide 2. the labelled cDNA will bind to the spots on the slides that contain complementary sequences 3. the spots that bind the labelled will then fluoresce under laser light in a piece of equipment known as a chip reader 4. the location and brightness of the fluorescent spots on the microarray identify the genes expressed in the test sample and their level of expression 5. Labelled spots on microarray identify expressed genes. 6. Relative fluorescent intensity indicates relative expression level

3 - Describe some of the benefits of being able to isolate and manipulate DNA (5)

o generate vaccines o create transgenic plants or organisms o diagnose genetic diseases o develop gene therapy o allow move to personalised medicine by identifying genetic changes leading to disease in an individual

32 - Describe what is meant by human genome sequencing (4)

• Advances in sequencing technology allows the ability to rapidly sequence genomes. • Original human genome was sequenced from samples combined from a number of individuals. • 100,000 Genome Project is now sequencing genomes of humans from various backgrounds, with rare disease or cancer to investigate basis of variation in humans and help to identify genetic basis of disease, explain individual differences in susceptibility to disease, response to drugs or reaction to environmental factors. • Identify single base pair differences (SNPs (small nucleotide polymorphisms)) between individuals.

45 - What happens to the products of PCR (2)

• Amplified DNA fragments are separated by gel electrophoresis • PCR product size equal to the amount of DNA between the primers.

14 - Describe the uses of cloned DNA (7)

• Clone and piece together genomic DNA to allow the mapping and sequencing of genes within the genome. • Identify changes in the genome that are associated with particular phenotypes, pathologies or diseases. • Characterise how genome is organised, location of repetitive DNA etc • Genetically engineer organisms by transferring genes between organisms. • Allow gene therapy or gene replacement treatment • Express large amounts of protein from isolated genes to allow study or production of new therapeutics (biologics). • Identify genes that are expressed in different cell types, i.e different cells in the same organism or genes expressed differentially in healthy and diseased organisms

9 = Describe how Agarose Gel Electrophresis works (1)

• DNA is visualised in gel by adding ethidium bromide which binds DNA and fluoresces when exposed to UV light.

53 - Describe the uses of the DNA Microarray (3)

• DNA microarrays can be used to directly compare gene expression in two samples • Can be used to compare gene expression in normal and diseased cells • The identification of the specific changes allow personalised therapeutic approaches

49 - Descibe what is meant by DNA microarrays (5)

• DNA microarrays or gene chips are modern devices which use nucleic acid hybridisation to rapidly measure which genes are expressed in a tissue sample. • Nucleic acid hybridisation occurs when complementary strands of DNA anneal to one another • Enables the ability to simultaneously identify all the genes expressed in a particular sample. • Uses an approach whereby all the mRNA from a sample is converted to cDNA, fluorescently labelled, denatured into single strands and used as probe. • This probe is applied to the array which contains spots where single stranded DNA from each gene in the genome is laid out in an array.

8 - Describe the process of how restriction enzymes can isolate genes (3) (Diagram)

• Different restriction enzymes recognize different nucleotide sequences. • The sequence forms a palindrome (sequence reads the same on both strands in 5'-3' direction) • Restriction enzymes cut in a characteristic cleavage to leave cohesive (sticky) or blunt ends

13 - Describe how cloned DNA is amplified (3)

• Individual recombinant plasmids can be taken up by E. coli bacteria. • Using the origin of replication (ori) 100-200 copies of the recombinant plasmid is generated in each bacteria. • As bacteria divide each one contains 100-200 copies of the recombinant plasmid

10 - Describe what is meant by recombinant DNA and how it is synthesised (4)

• Large amounts of individual DNA fragments are needed to allow further study, manipulation or to develop therapeutics. • Use bacteria or yeast to replicate and amplify individual DNA fragments • Need mechanism to introduce DNA into host organism • Use plasmids (circular DNA molecules) as 'cloning vectors' to carry DNA fragments into host

15 - Describe the issues when cloning eukaryotic genes (5)

• Most eukaryotic gene contain introns. • The introns can be large so eukaryote genes may span 10-100kb • If we wish to express protein or characterise gene function we cannot use genomic DNA as contains introns. • Need to use mRNA where the introns are removed and convert this back to DNA (reverse transcription) • Different cells express different genes, i.e., not all cells express same genes. Harvesting mRNA from different cells also allows us to discover which genes are expressed by which cells and how this changes in disease.

46 - Describe the uses of PCR (6)

• PCR can amplify DNA fragments from tens of bases in length to 5kb. • Wide variety of uses. • Can amplify whole genes or parts of genes from genomic DNA or cDNA for use in cloning and molecular analysis. • Can amplify specific regions of DNA from variety of samples, e.g. specific gene in different individuals to perform genetic diagnosis. • Can amplify DNA from sources where DNA is limited, e.g. blood spots, fossils • Identify victims in natural disasters, major accidents, e.g. World Trade Center

33 - Describe what is meant by the polymerase chain reaction and what are the processes (5)

• PCR is an alternative method to make many copies (clones) of specific DNA fragments without need to use cloning vector • Very sensitive, able to amplify DNA fragments from very small amounts of DNA, 1picogramme (10-12g) of DNA is sufficient, but possible to use as little as 1femtogramme (10-15g). • Many applications, e.g. in diagnostics and forensics • Can amplify sections of genome known to be affected in disease to detect carriers of human genetic disease • Can amplify DNA from single human hair to identify individuals

47 - Describe the applications of PCR (6)

• PCR is routinely used in screening of mutations involved in genetic disorders. • PCR is a key diagnostic methodology for the detection and identification of bacteria and viruses in humans • Identify pathogenic bacteria in contaminated food. • PCR techniques are used to analyse samples from single cells. • PCR used in forensics to identify individuals from body fluids left at a crime scene or in paternity testing. • Used to check and confirm DNA constructs or transgenic animals.

11 - Describe why plasmids are used to form recombinant DNA (3) (Diagram(

• Plasmids contain an origin of replication that allows replication independently of the bacterial chromosome. • This allows high copy numbers of plasmids, 100-200, to be maintained in each bacteria. • Plasmids also contain antibiotic (AB) resistance genes that allows the selective growth of bacteria that contain plasmids.

4 - Describe the benefits of gene cloning (3)

• Recombinant DNA technology (or Gene Cloning) allows the isolation and manipulation of DNA. • Made possible by using methods to isolate sections of DNA and then create multiple copies of these sections of DNA. • Known as Gene Cloning because copies (or clones) made of unique pieces of DNA.

7 - Describe the role of restriction enzymes and why it is important (3)

• Restriction enzymes function as 'molecular scissors' to cut DNA • Restriction enzymes recognize specific nucleotide sequences in the DNA and cuts both strands of the sugar-phosphate backbone • First identified in bacteria where they protect against viral infection by cutting (restricting) viral DNA

28 - Describe how sequencing technologies have advanced (4)

• Sequencing technologies have advanced allowing ability to sequence larger amounts of DNA quicker. • Original radioactive dideoxy sequencing on gels average read 200-400bp per reaction and took several days to get sequence • Fluorescent capillary dideoxy sequencing average read 800-1000bp, takes hours to get sequence, easy to sequence many fragments can generate 10-20,000bp per hour • Next generation sequencing (NGS) uses sequential addition of nucleotides on microchips, short reads of 50- 200bp but very rapid, can sequence thousands to millions of DNA fragments per day to generate over 200- 600Mbp per day.

34 - Describe the process of the polymerase chain reaction (5)

• The PCR uses repeated cycles of targeted DNA replication to amplify large amounts of a specific DNA fragment. • PCR involves three steps - denaturation, primer annealing, and extension. • These are repeated over and over using a thermocycler to amplify the DNA exponentially. • The number of copies of the fragment is doubled in each cycle, 2,4,8,16, 32, 64, 128, 256 etc • The new strands along with the old strand serve as templates in the next cycle. • After 30 cycles 230 copies are generated, approx 109 copies

27 - Describe how a fluorescent pattern is made from automatic DNA sequencing (5) (Diagram)

• The fluorescent label on each ddNTP has a different wavelength • As the fragments move down the gel it passes a laser, smaller fragments move faster • The laser light excites the fluorescent tag on each fragment as it passes • The wavelength of the fluorescence is read as the fragment passes, and the information is recorded. • The fluorescence pattern produced shows the sequence of the DNA

16 - Describe the importance of cDNA in eukaryotic DNA replication and how this can happen (6)

• To express eukaryotic proteins or identify genes expressed in a particular cell type need to make DNA copies of the transcribed mRNA • mRNA is produced when genes are transcribed • mRNA does not contain introns • but cannot clone RNA, only DNA • Complementary DNA (cDNA) is a DNA copy of a mRNA, produced using an enzyme called reverse transcriptase. • cDNA libraries contain complementary DNA copies of the mRNAs present in a cell population and represent the genes being expressed in the population from where the mRNA is harvested. • cDNAs are used as the introns are removed and the clone size is smaller

20 - Describe what DNA sequencing is and how this can occur (5)

• Uses DNA polymerase to copy single stranded DNA • Makes use of 'dideoxy' nucleotides which interrupt the ability of DNA polymerase to copy DNA • DNA polymerase cannot continue to extend the chain of nucleotides after incorporation of a dideoxynucleotide • Method known as the dideoxynucleotide chaintermination sequencing • This method can be automated and allows rapid sequencing of large amounts of DNA

26 - Explain how automatic DNA sequencing occurs (5) (Diagram)

• When DNA is replicated by DNA polymerase in presence of dCTP and small amount of ddCTP some fragments end prematurely. The length of the fragments depends on when ddCTP is incorporated. • Automated DNA sequencers use a single reaction in which all four ddNTPs are included together with dNTPs • Each ddNTP is labeled with a unique fluorescent marker • We can identify which ddNTP was added at the end of each fragment by identifying its fluorescent colour • The DNA sequence fragments are separated on a capillary gel