BIO230 Molecular Biology

1. Cut DNA

• Restriction enzymes, cut DNA at specific sequences
• Sequences normally palindromic
• Causes double stranded break
  • Sticky end from staggered break
  • Blunt end when cut in the middle

2. Joining DNA

• The H-bonds between the sticky ends are unstable
• T4 DNA ligase is used to repair the single stranded breaks in DNA
• Forms covalent phosphodiester bond between 3'OH and 5'phosphate
• Required ATP cofactor for energy

3. Introducing DNA into hosts

• Using Transformation
• uptake of foreign DNA causing a genetic alteration in the host cell or body
• Done by Chemical treatment, electroporation or conjugation

4. Copy the Clone

• Use vectors to make sure the introduced DNA is replicated and partitioned into daughter cells
• E.g. Plasmids
  • Have an origin of replication
  • May be transmissable
  • Small
  • Carrry selectable genes e.g. antibiotic resistance
  • High copy number
  • Non-self transmissable
  • E.g pUC18
  • Have multiple cloning sites

5. Cloning into the plasmid 

• Cut plasmid with restriction enzyme, the restriction site is cut in half, cut the desired DNA fragment with the same restriction enzyme/ one that gives complementory sticky ends. To prevent the vector recirciling without the wanted fragment add alakaline phosphotase. 

6. Selecting the vectors with the taken up DNA

• Use Blue/White selection
  • Insert wanted fragment into lac Z gene. When the gene is broken, it cannot make the enzyme B-galactosidase, which breaks down lactose to glucose and galactose. If the chromogenic substance, x-gal is added and the enzyme is made, it will go blue, if the gene is broken, it will stay white. The white plasmids can then be selected.

7. Analysing recombinants

• Use gel electrophoresis
  • DNA has a negative charge, so will move along a gel when an electric field is applied 
  • DNA will move at different speeds depending on the fragments size.
  • Gel can be agrose or polyacramide
  • Agrose gel will vary in concentrations depending on the size of the fragments being tested, general purpose 0.7%, larger fragments are placed in 0.2-0.5%, smaller fragments placed in 0.7-1%

• Amplification of specific DNA sequences
• In vitro DNA synthesis 
• DNA Taq polymerase, primers and free nucleotides are used

Cycle 1

• Heat separates strands (94)
• Cool to allow primers to anneal (55) and extension (74) 
• Complementory strands synthesised
• 2 new DNA fragments are made, become template strands for second cycle

Cycle 2

• Repeat heating and cooling procsseses of cycle 1
• 4 new strands synthesised
• 2 are the correct length, with primers incorporated

Cycle 3

• Form 2 correct lengh double stranded molecules, then replicated logarithimically 

A DNA librbary is a collection of clones each containing a different piece of cloned DNA

Genomic library

• Represents teh entire genome of an organism

cDNA library

• Represents RNA sequences 
• Only genes which have been expressed in a particular starting material

Uses

• Locating genes
• Whole genome shotgun sequencing
• Identifying genes expressed

Problems

• Eukaryotic genomes are very large and bacteria cannot read eukaryotic mRNA

Uses hybridisation of a DNA probe

Nylon folter disk is placed onto the surface of the agar plate

This produces a mirror image of the plate

The DNA is then released by lysis and bound to to the filter

The probe hybridises the clones which contain the wanted DNA

This is then visualised by autoradiography

Library formed from mRNA

Does not contain introns

cDNA prepared from mRNA can be expressed in bacteria 

Composition of the library will vary as diffferent patterns of gene expression are found in different tissues

Making a cDNA library

• Isolate mRNA from starting tissue
• Synthesis cDNA using polyT primers and reverse transcriptase
• Clone cDNA into vector
• Then screen library for wanted gene

Properties

• Genes vary in abundance, no introns

Homology

• There are a core set of genes at the origin of evolutionary diversification
• There are many more molecular interactions that cntrol cell biology that are shared by a huge variety of organisms 
• Studying a protein in a model organism allows us to infer or predict its function in another less environmentally used organism

Attributes                                                                                                                                          

• Small and simple
• Rapid life cycle
• Easy to grow and propogate
• Good yield of progeny
• genetic and physical maps known
• small genome
• clear mutant phenotypes

95% of the human genes are alternatively spliced, so it is not the genes within the organism but how they are transcribed.

A gene encodes for one or more transcripts that can function as an RNA or be transcribed into one or more proteins

1. Northern analysis of genes

• Used as evidence for gene models
• Total RNA is separated via gel electrophoresis
• It is then transferred onto a nitrocellulose membrane
• This is then probed using a labelled DNA probe corresponding to the gene of interest
• If the gene is being expressed then a bond will appear on the blot
• Can deduce the size and abundance of the gene

2. Reverse transcriptase PCR

• Used to analyse expression of single genes
• cDNA synthesised using mRNA and reverse transcriptase
• PCR amplification using primers
• Gel electrophoresis to find gene of interest
• Technically straight forward, fast, semi-quantitive and senstive for small sample use

3. Quantitive PCR

• Maplification is coupled with to the generation of a fluorescent molecule that can be accurately measured.
• Done one gene at a time

• Allows for all of the genes of an organism to be analysed simultaneously 
• Consists of tiny amounts of a large number of single stranded DNA fragments representing different genes fixed to a glass slide in a grid

Procedure

• mRNA's from the cell of interest isolated
• Used as templates to make cDNA
• cDNA's are labelled with different coloured flourescent molecules
• cDNA then hybridises to a DNA microarray

Problem

• Difficult to detect genes that are only slightly induced/ repressed or that are only expressed in a few cells in a tissue
• qPCR used to validate results

Uses

• Idetification of gene networks, finding response genes, predicting clinical responses

• Sequence cDNA from different tissues of sifferent embryonic development in order to discover which genes are expressed

Method

• Isolate mRNA
• Convert to cDNA
• Sequence products using Illumina sequencing
• The number of reads from each gene indicates its relative expression in the sample

RNA in-situ hybridisation

• Works like a northern blot but directlyon a tissue, allowing the exact site of gene expression to be determined

1. Forward genetics

• Identifying the genes via the phenotype they cause
• Use saturation mutagenesis
• Disable a gene then observe the consequences in the cell

Generating mutations

• Random chemical mutagenesis
• Random transposon tagging mutagenesis
• Imprecise excision of a transposon
• Targeted mutations by homologous recombination

Reverse genetics

• Starting with a gene and working out the phenotype is causes, use loss of funtion mutation
• Can use sequence homology, BLAST to infer a function to a gene 

• Stands for Basic local Alignment Search Tool
• Finds regions of local similarity between sequences 
• The programme compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches
• Can be used to infer functional or evolutionary realtionships between sequences as well as help identify members of gene families

Process

• Domain detection 
• Detection of homologous proteins

• Powerful method for gene discover and analysis
• Based on making cross-species comparisons to identify genes or proteins and gain insight into their function
• As more species are sequenced it enables us to identify DNA sequences/ proteins that are evolutionary conserved

Synteny

• Refers to segements of chromosomes from different species sharing the same linear organisation of genes 
• Regions are referred to as synetic

Targeted mutagenesis

• Generating a small deletion by mobilising a transposon 
• Knock-out by homologous recombination
• Generates DNA double stranded breaks for mutagenesis
• Gene knockout by RNAi

• Stands for Clustered Regulary Interspaced Short Palindromic Repeats CRISPR associated nuclease 9
• Genomic manipulation by CRISPR-Cas9 completely degrades the protein and does not effect untargeted protiens
• Endogenous to bacteria and archaea
• Cas 9 enzyme catalyses the formation of ds breaks
• This is then repaired, creating frame shift insertions

1. Forensic DNA profiling

• DNA profile is uniique to the indicvidual 
• Use minisatellite DNA
  • Region of 20kb
  • Composed of short repeat sequences (20b)
  • Repeat sequences vary more between individuals than unique sequences
  • Repeat sequences are highly herterozygous
  • They also have multiple alleles and a high mutation rate 

2. PCR

• Detects microsatellite locus
• Amplifies the microsatellite using locus specific primers
• Primers labelled with a flurophone
• Determine size of PCR products by capillary electrophoresis
• DNA for PCR can be extracted from very small samples

3. DNA fingerprinting

• Samples obtianed via non-intrusive methods, e.g. from hair or faeces
• Primers used to amplify microsatellite in humans, are the same primers used for chimps 
• Can be used to find mating patterns

4. Microsatellite profile match

• Curently eleven loci are amplified in humans to identify an individual
• 10 STR sequences (short tandem repeat)
• 1 sex-specific sequence- Amelogenin from tooth enamel
• Each STR is on a different chromosome

• Used to find inherited disorders
• Blood samples amnoicentisis ad foetal cells can be used for genetic tests

1. Huntington Disease

• Slowly progressive brain disorder
• Mental disturbance
• Physical deterioration
• Look for CAG repeats
• Healthy individual will have few CAG repeats
• Fluorescent PCR used to detect CAG repeats
• Molecular beacon used, only fluoresces when bound to the template

2. Cystic Fibrosis

• Affects many organs
• CFTR gene on chromosome 7 affected, codes for chloride membrane channels 
• Range of mutations correlate with severity of symptoms 
• Recessive, 75% of those affects carry F508 mutation

1. Multiplexing

• Detetcing a range of mutations 
• One pair of PCR primers to amplify affected gene 
• Molecular beacon for each mutation and for each wild type
• Each molecular beacon is labelled with a different fluorophore 
• Can detect any of a range of mutations
• Can distinguish different mutations

• Readily detectable section of DNA
• Shows differences between individual and another

RFLP- Restriction fragment length polymorphism- detects different locations of restriction sites

Microsatellite locus

SNP

Marker assisted selection

• More acceptable than GM
• Locate markers that lie close to genes controlling a key trait 
• Select progeny on the basis of the marker rather than the trait
• Useful when
  • Trait is difficult/expensive to measure
  • Pyramiding
  • When the trait involved takes a long time before it can be measured e.g. fruit quality in apples

1. Pyramiding resistance genes

• Barley Yellow mosaic virus
  • Has a range of related virusues 
  • Need number of resistances
  • Marker ***isted breeding used

2. Global Food security

• Demand for food will grow by 40% by 2030 and 70% by 2050
• primary production needs to be increased 
• Wild relatives of crops are a source of useful triats
• Rice
  • Could *** PSTOL1 gene which gives phosphorus starvation tolerance 

3. Species idnetification

• Identify and protect endangered species
• Applicable to all life stages
• Don't need complete animal 
• Short DNA sequence froma standard part of the genome used
• Use part of the mitochondrial genome
• Can identify species using sequence comparison of mitochondrial cytochromic oxidase 1
• E.g. whale conservation 
  • mDNA sequencing used to moniter whaling ban
  • Can be used to geographical location of the kill

• Study of genetic material recovered directly from the environment 
• Relies on high throughput sequencing

Bacteria are hard to culture from the environment, 16s RNA used

16s RNA

• Forms a part of the small subnit of the bacterial 70s ribosome
• Highly conserved 
• Constantly regions
  • For universal primers
• Variable regions
  • Distinguish between species

Use

• Extract bacteria from environment, extract DNA and shotgun clone it
• Sequence the clones and compare with DNA databases and identify organisms and any new genes

Nanopore

• Single molecule sequencing in real time
• DNA is driven through a nanopore 
• Fluctuations in conductance indicates base

High throughput sequencing

• Next generation sequencing 
• 454 pyrosequencing
• addition of base release PPi
• Detect PPi to determine which base is added

• Cancer 
  • Whole genome or exome can be sequenced 
• Inherited disease 
  • Identifying the cause of rare genetic diseases 
• Epidermology
  • Control outbreak of MRSA
  • Determine the antibiotic resistance profile of TB

Breast cancer

• Identified 10 different types
• 10 year survival rates vary from 40-90% 
• Potential to tailor treatements to tumour type