Trimester Revision

Question 1

What is bioinformatics?

Answer 1

Analysing and predicting information using the structure, function, regulation and organisation of the genome and proteome.

Question 2

What is an example of first generation DNA sequencing?

Answer 2

Sanger sequencing. Bases are assigned different coloured dye to read the DNA sequence. A cheap but slow method that uses terminator bases.

Question 3

What is second generation sequencing?

Answer 3

High throughput method that gives millions of short DNA segments that will be amplified and reassembled.

Question 4

What is third generation sequencing?

Answer 4

DNA up to 10,000 bases can be read in a single run without amplification.

Question 5

What are molecular diagnostics?

Answer 5

Lab techniques that analyse the health of an individual. Can examine the molecular basis of disease to draw a medical diagnosis.

Question 6

What can molecular diagnostics be used for?

Answer 6

Accurate, detailed, reproducible identification of the mechanisms and causes of disease as well as detection, diagnosis, prognosis and monitoring response to therapy.

1. Biomarker screening and monitoring
2. Pathogen detection and quantification
3. Genotyping to identify mutation and forensic evidence

Question 7

`PCR steps:

Answer 7

1. Denaturation: (94 C for 15 sec-2min)
2. Annealing (40-60 C, primers anneal)
3. Extension (70-74 C, polymerase adds bases).

Components= template, primers, polymerase and buffer

Question 8

What is a Tm?

Answer 8

The annealing temperature of a primer. If temperature of PCR is too high above Tm, primers can’t attach. primers should be within 5 degrees of each other and should not have complementary sequences to ensure no primer dimers are formed.

Question 9

RT-PCR:

Answer 9

PCR for RNA. RNA will form cDNA using reverse transcriptase.

Question 10

What are the levels of structure a protein?

Answer 10

Primary: amino acid sequence
Secondary: alpha helices and beta sheets
Tertiary: interactions between the secondary structures
Quaternary: interaction between two or more tertiary structures

Question 11

Does mRNA have introns?

Answer 11

NO, as it has been processed.

Question 12

How is mRNA translated?

Answer 12

Using tRNA and its anticodons that correspond to codons on mRNA and bring in amino acid.

Question 13

What are the types of DNA ‘variants’ in humans?

Answer 13

1. Single nucleotide polymorphism
2. Insertion and deletion
3. Structural variants (DNA >1000bp affected)
4. Repeat variations (tandem repeats accounting for 45% of the genome)

Question 14

Where do restriction enzymes come from?

Answer 14

Phage Lambda

Question 15

What is unique about restriction enzyme recognition sites?

Answer 15

They are palindromes.

Question 16

How does gel electrophoresis work and what is it used for? How is it limited?

Answer 16

Seperates fragments of DNA by size using an electric field and a gel matrix. When looking to identify a mutation we must know which mutation is present and it must be compatible with a restriction enzyme.

Question 17

How can single nucleotide polymorphisms occur?

Answer 17

Through silent mutation (no change in amino acid produced), missense mutation ( the amino acid changes) and nonsense mutations (the amino acid becomes a stop codon)

Question 18

How can SNP’s be detected ?

Answer 18

By ‘chemical cleavage of mismatch’, detecting mutations by comparing DNA to its wild type.

Question 19

What is capillary zone electrophoresis and how does it work?

Answer 19

This is a technique that seperates molecules based on their mass/charge ratio. Electro-osmotic flow from a high voltage is applied to the capillary, causing cations of the analytes to move to the cathode. Velocities dtermined by the mass/charge ratio ( a smaller molecule with a higher charge will move faster)

Question 20

What is capillary gel electrophoresis and how does it work?

Answer 20

This is used when charge of compounds doesn’t vary with size much. Capillaries will be coated to prevent electroosmotic flow and a polymer inside acts as a sieve for separation by size. This is done at 60 C.

Question 21

What happens during Sanger sequencing?

Answer 21

Bases labelled with fluorophores will be added to the DNA- these will terminate the DNA chain in random places, producing fragments of different length. This can be observed on an electropherogram, and the results can be sequenced (and separated by gel electrophoresis).

Question 22

What is shotgun sequencing?

Answer 22

This a technique used for large amounts of DNA in which fragments will be generated by restriction enzymes and cloned. The clones will be sequenced using a universal primer and be assembled into contigs by computer programs that can align ends of different reads to form a single sequence.

Question 23

How does HiSeq X Ten sequencing compare to the techniques used to sequence the first human genome?

Answer 23

This technology can sequence 45 genomes in 1 day for $1000 each, whereas the first genome took 15 years and $3 billion. The system uses ‘massively parallel sequencing’ that sequences clusters instead of single strands of DNA. Sequencing can be done by synthesis ( as DNA is formed ) or by ligation ( forming clusters of identical DNA ). The sequence can be read from an immobilised strand on a chip.

Question 24

How does Next Gen sequencing work?

Answer 24

Uses fluorescent dyes similar to Sanger, but doesn’t use capillary electrophoresis. Instead we use array based sequencing that processes millions of reactions at the same time. Has lower accuracy and shorter read lengths, which is counteracted by reading many copies of the same fragment.

Question 25

What are the three common steps in all Next Gen Sequencing techniques?

Answer 25

1. Library preparation: a library is created by fragmenting DNA with restriction enzymes and joining the fragments to labelled linkers (adaptors) 2. Amplification of the library : by clonal amplification inside bacteria and using PCR. Resin beads will be added and DNA on the beads are complementary to sequences on the linkers , allowing binding to the beads. The fragments captured by the beads are copied by PCR. 3. Sequencing: amplified DNA is sequenced by synthesis or ligation. Remaining beads put into wells on a sequencing plate with enzyme beads containing DNA polymerase and primer for sequencing. The enzyme and primer will attach to DNA on the beads and nucleotides will be added to the wells in waves of one base type at a time. Light will be produced and recorded.

Question 26

How does pyrosequencing (Roche 454) work?

Answer 26

The 454 plate image is recorded by light given out when bases are added. More intense light means more nucleotides of the same base have been added. Does 500 megabases per run but is not reliable.

Question 27

How does Illumina sequencing work and compare to Roche 454?

Answer 27

Illumina: cheaper, 1000 x max output over Roche, takes longer Allows multiplexing with indexes. 1. Prepare library 2. Load library in flow cell and hybridize fragments to surface. Bound fragments amplified into clonal cluster by bridge amplification. 3. A sequencing cycle creates a digital image of the clusters 4. Alignment and data analysis is done using a computer and bioinformatics.

Question 28

How does Applied Biosystems SOLiD work?

Answer 28

Does sequencing by ligation only, does not use polymerase, very accurate as it uses short fragments. Uses emulsion PCR using DNA bound to beads and DNA will covalently bind to surface of a slide using adaptors. Probes will bind to complimentary DNA, releasing fluorescent dye when they bind. Dinucleotides are added at each point which will be aligned in sequencing.

Question 29

What are the applications of Next Gen sequencing?

Answer 29

- whole genome sequencing, comparative genomics, transcriptomics (RNA sequencing), amplicon (bacteria, fungi and virus) and shotgun meta genomics

Question 30

Reading DNA for use:

Answer 30

DNA section read 100 times to identify read errors in whole genome sequencing. The reads will be mapped to a reference sequence to identify SNPs. This info can then be used for comparative genomics and for sequencing the whole genome. individual reads are put together to create a map of the whole genome.

Question 31

What is transcriptomics?

Answer 31

sequencing of RNA to see which genes are transcriptionally active. The RNA will be converted to cDNA for the library. Used to study disease, diagnostics and treatments. Quantitative → green means more translation and red is less

Question 32

What is metagenomics?

Answer 32

studies genetic material from the environment. Not all bacteria can be cultured. DNA from an environmental sample is extracted, cloned into a vector and transformed into E.coli. The transformed cells are screened for novel features. Contains DNA sequences for genes of microbial populations.

Question 33

What is amplicon metagenomics?

Answer 33

looks at the gut microbiome. Looks at predetermined marker genes (short sequences). Very cheap and high throughput.

Question 34

What is shotgun metagenomics?

Answer 34

Sequences all DNA in a mixed sample. Don't have to sort. More accurate for functional gene analysis than amplicons. More expensive and not high throughput. DNA is extracted, fragmented, cloned into vectors and transformed into bacteria. Library is sequenced and contiguous fragments are assembled.

Question 35

What is karyotyping?

Answer 35

Arrangement of a metaphase spread of chromosomes in decreasing length to view size, shape and number. Giesma stain used that attaches to G bands. Centromere position is on the dotted line.

Question 36

Reading a chromosome position:

Answer 36

8q22.1 - chromosome 8, q arm, band 22.1

Question 37

What is cytogenetics:

Answer 37

Studies how DNA affects cells, looking at whole chromosomes and their bands, NOT DNA SEQUENCES. Compare chromosomes and using molecular labelling.

Question 38

How do chromosomal abnormalities occur?

Answer 38

Abnormalities come from errors in division, for example non disjunction during anaphase I or anaphase II. Nondisjunction increases with maternal age and exposure to certain drugs, both factors that increase risk of chromosome abnormalities.

Question 39

What are numerical and structural abnormalities?

Answer 39

Numerical : extra or missing chromosome Structural: translocation (part of chromosome transferred to another), inversion (part of chromosome broken off turned upside down and reattached), deletion (portion is missing or deleted), duplication (portion of chromosome duplicated), ring formation (part of chromosome breaks off, forms ring)

Question 40

What is a structural aberration?

Answer 40

two types: loss of genetic material or rearrangement in location of genetic material (del, dup, inv, ring, transl)

Question 41

How does FISH work?

Answer 41

labelled fluorescent probes bind to specific denatured DNA target sequences then can identify where the probe is located.

Question 42

What is cross species colour banding?

Answer 42

allows better resolution, more sites can be identified. We hybridise human chromosome with part of chromosomes of two Gibbon species.

Question 43

What is a centromere/locus specific mFISH?

Answer 43

mFISH doesn't work on centromeres, probes used are centromeric probes. Distinguishes all centromeric regions apart from 13 and 21. Fast and cheap and used in prenatal, postnatal diagnosis

Question 44

What is the template strand called and in what direction is it read?

Answer 44

It can also be called the antisense strand and is read in the 3" to 5"

Question 45

What enzyme synthesizes RNA?

Answer 45

RNA polymerase II

Question 46

What is chemical cleavage of mismatch?

Answer 46

Detects unknown mutations + anything not identical. If mutations are small, it can form a heteroduplex with a complementary strand that doesn't have the mutation. Hydroxylamine will cut cytosine, while potassium permanganate reacts with thymine. A TC mismatch will be closed by piperidine, and fragments can be analysed using fluorescent labels and PCR.

Question 47

What is a single strand conformation polymorphism?

Answer 47

Uses electrophoresis to detect all mutations and hence their conformational differences in single stranded DNA. Denatures DNA strands so that they can form their conformation. Different confirmations will cause DNA to travel differently in electrophoresis.

Question 48

What is massively parallel sequencing?

Answer 48

Instead of analysing single strands, many clusters are formed at the same time

Question 49

What does a Giesma stain attach to?

Answer 49

AT rich regions (G bands)

Question 50

What can cytogenetics be used for?

Answer 50

cancer detection, prenatal diagnostics, abnormal chromosome structure and number

Question 51

Will chromosome abnormalities that originated from the gametes be present in every body cell?

Answer 51

Yes.

Question 52

What are STRs?

Answer 52

Short tandem repeats. Non coding regions that are highly variable and used in DNA fingerprinting.

Question 53

Y chromosome STR use:

Answer 53

can be used in paternity tests for sons and identifying male relatives. All paternal relatives share a Y marker. Y STR loci is haploid. 98% of violent crime done by men, more than 100 Y linked STR loci discovered that live on the non recombinant portion of the Y chromosome (reliable paternity test)

Question 54

Mitochondrial DNA use:

Answer 54

inherited from the mother, closed circular DNA, does not undergo recombination, codes for 13 respiratory system proteins, accumulates mutations fast. Can trace human migration. can have diseases that are associated with organ failure and cell death. Copy Number Variations are tested and determined to be pathogenic, likely pathogenic, variant of uncertain significance, likely benign, benign.

Question 55

What is epigenetics?

Answer 55

Inheritable changes in DNA that affect gene expression in response to the environment (temp, radiation, food, drugs) but do not affect the DNA sequence. DNA methylation and post translational modification of histones.

Question 56

What does DNA methylation do?

Answer 56

lock genes in 'off' position. Cytosine bases are methylated and converted to 5-methylcytosine by DNA methyltransferase.

Question 57

What happens in post translational modification of histones?

Answer 57

HIstones can be acetylated (activates gene expression), methylated (can allow or repress transcription) and phosphorylated (relaxes chromatin). Histone deacetylases remove acetyl groups. Histone acetyltransferases add acetyl groups. Histone Lysine methyl transferases add methyl groups to lysine's. Histone demethylases remove methyl groups from lysine's. All modifications can be added or removed in response to external signals

Question 58

What are imprinted genes?

Answer 58

have their expression inherited from a parent, located in clusters, each having a control region to regulate their expression. DNA methylation controls expression of these genes.

Question 59

Methylation in tumours:

Answer 59

Tumour cells have abnormal methylation (CpGs becoming unmethylated and CpG islands being methylated.) Losing methylation may cause unstable chromatin and activate oncogenes, may even cause overexpression of oncogenes. Gaining methylation can inactivate tumour suppressor genes.

Question 60

What is bisulfite sequencing?

Answer 60

studies loci specific DNA methylation, based on converting genomic DNA using sodium bisulfite. Methylated regions become unmethylated. Unmethylated cytosine residues are converted to uracil while 5-methylcytosine is unaffected. After PCR, uracil is converted to thymine and methylation status can be determined by PCR sequencing or cloning sequencing.

Question 61

What is MEDIP (methylated DNA immunoprecipitation)?

Answer 61

allows genome wide study of DNA methylation using a microarray. Using an antibody to capture methylated fragments which will then cause them to precipitate. Precipitated, enriched DNA can be labeled fluorescently and non precipitated DNA can be labeled with a different colour. The fragments can rebind and the colour that shows up demonstrates how much DNA is methylated

Question 62

Pharmacogenetics:

Answer 62

study of inherited differences due to single allele variation in gene

Question 63

Pharmacogenomics:

Answer 63

study of alterations to multiple loci across the genome

Question 64

Candidate gene approach:

Answer 64

tests if an allele or set of alleles are more frequent in patients with a better or worse drug response. Smaller sample size requirement and lower cost but can miss the causative SNP and requires prior knowledge of gene function.

Question 65

Candidate pathway-based studies:

Answer 65

investigate genes with respect to the pharmacology of a drug, disease or biological pathway. Can evaluate more genes in a signaling pathway that a drug is assumed to have an effect on but still requires prior knowledge of the function of alleles.

Question 66

Genome Wide Association Studies (GWAS):

Answer 66

studies genetic variation in the entire human genome using high density SNP array to identify genes that contribute to diseases. No prior knowledge of drug response mechanism or gene involved is needed but a large sample set is required, false positives are common, the study is expensive and analysis/management of the data is difficult.

Question 67

Pharmacokinetics:

Answer 67

concentration of active drug in body over time, will at first increase then decrease. Examines absorption (route of admin, dosage and absorption rate), distribution (transport of drugs and distribution to tissues), metabolism (drugs altered by enzymes, forming metabolites, this occurring in the liver) and elimination of the drug by enzymes (usually done by the kidney), membrane drug transporters and plasma proteins.

Question 68

Pharmocodynamics:

Answer 68

concentration of a drug vs the effect. Looks at at target proteins for a drug - the drug will be aimed at a protein in the body. Hence PK/PD gives us the effect of a drug vs time.

Question 69

What is a probe?

Answer 69

Binds specifically to analyte (target) and will release a detectable signal or change. single stranded RNA or DNA that is complementary to and hybridises to a sequence of interest by hydrogen bonds. 100-1000 bp and labelled. Used in in situ hybridisation, dot blots, colony screening, DNA profiling, Southern Blots, northern blots and diagnostic tests.

Question 70

Factors that affect hybridisation

Answer 70

Probe length, base composition, chemical environment, temperature

Question 71

Steps before hybridisation:

Answer 71

1. Isolate sample from cells 2. Check sample for quality 3. Treatment of the sample 4. Separation of the sample 5. Immobilisation of the nucleic acids on a support

Question 72

Stringency:

Answer 72

specificity of which a target sequence is hybridised to a probe. With high stringency, sequences must be perfectly complementary to hybridise. This can be achieved with a high temp and low salt. Low stringency comes from low temp and high salt.

Question 73

Steps in a Southern Blot

Answer 73

transferring DNA from agarose onto a membrane. Detects changes in DNA between samples. 1. Isolate and purify DNA 2. Restriction digest 3. Electrophoresis of fragments 4. Transfer DNA to membrane 5. Baking or UV cross link nucleic acid to membrane 6. Hybridisation with probes 7. Probe detection

Question 74

Northern Blot steps

Answer 74

transfer of RNA from agarose gel to a membrane. Detects changes in gene expression 1. Isolate RNA 2. Electrophoresis with formaldehyde 3. Transfer to membrane 4. Baking or UV cross link nucleic acids 5. Hybridise and detect the probe

Question 75

What is a Western blot?

Answer 75

transfer of protein from polyacrylamide gel to membrane. This uses antibodies instead of probes.

Question 76

What is FISH?

Answer 76

DNA probes are chemically labelled and detected with fluorescent antibodies. Cells lysed under alkaline conditions to denature DNA. Probes are bound to fluorescent molecules. Can visualise expression of a gene, co-expression of genes, can give medical diagnosis', detect abnormal karyotypes, progression or remission of cancers.

Question 77

What is Huntington's?

Answer 77

a neurodegenerative inherited disorder, caused by autosomal dominant mutation in the gene Huntingtin that causes mutated proteins to accumulate in the brain. There will be increased repeats of the sequence CAG. Causes uncontrolled movement, emotional issues and loss of cognition.

Question 78

What are BRCA genes?

Answer 78

BRCA 1 and 2 stabilise the genome, acting as tumour suppressor genes in all humans and code for breast cancer type 1 and 2 susceptibility proteins. Can diagnose early breast cancer. Repair damaged DNA and destroy cells if repair cannot be done.

Question 79

How does CRISPR work?

Answer 79

uses Cas9 protein, an endonuclease to cut DNA. It is complexed with a synthetic guide RNA which will allow the genome to be cut at a specific site, removing or adding genes.

Question 80

How can the double break in DNA from CRISPR be repaired?

Answer 80

1. NHEJ- non-homologous end joining: broken ends are ligated without need for a homologous template. Can cause inaccurate repairs and loss of nucleotides, may even cause tumours. 2. HDR- homology-directed repair: Usually homologous recombination, can be used when a homologous piece of DNA is in the nucleus to replace it.

Question 81

How are proteins processed for proteomic analysis?

Answer 81

Fresh or frozen tissue need protein removed from the raw tissue, while formalin fixed and paraffin embedded tissue need heat induced antigen retrieval for proteomic analyses.

Question 82

Reverse Transcription PCR

Answer 82

reverse transcriptase copies RNA into cDNA that can then be amplified and sequenced with PCR. This is due to many viruses, like SARS being an RNA virus.

Question 83

Real time PCR:

Answer 83

accumulation of DNA is measured after each cycle as opposed to conventional PCR that measures at the end of the reaction. Detection of PCR products is done with fluorescent molecules that release fluorophores when amplified. Measured fluorescence at each cycle relates to the amount of DNA. Allows determination of the initial number of copies of template DNA and can be evaluated without electrophoresis. As it is done in a closed tube, contamination is reduced.

Question 84

Cystic Fibrosis

Answer 84

inherited disease that affects lungs. Mutation occurs in both copies of the gene for the CFTR protein (eg delta F508 deletion)

Question 85

PCR:

Answer 85

Denaturation at 94 C for 15sec-2min Annealing at 40-60 C Extension at 70-74 C involves thermostable DNA polymerase, buffer, magnesium, nucleotides, primers, target DNA

Question 86

What are CpG islands?

Answer 86

These are usually unmethylated. Are regions of DNA with high frequency of CpGs, often associated with genes and gene promoters

Question 87

VNTR (variable number tandem repeats):

Answer 87

are regions of DNA where motifs (sequences) are repeated directly adjacent to each other

Question 88

How many STR alleles do we use in Aus?

Answer 88

18 alleles are used in the National Criminal Investigation Database in Australia. If 13 alleles match = 1 in 1.5 billion chance that this is not the suspect.