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125 Cards in this Set

  • Front
  • Back

How can we characterise genes within a genome?

Full characterisation of genes within the genomecan be obtained by sequencing DNA clones

How can we predict the sequence of amino acids contained in the protein a gene encodes?

This provides the sequence of bases which canbe used to predict the sequence of amino acidscontained in the protein that the gene encodes.

What does this allow us to do?

This allows us to determine some properties ofthe gene product.

What information does the DNA sequence also give?

DNA sequence also gives information about how the genome is organised e.g. regulatory regions (promotors/enhancers), satellite DNA,telomeres, centromeres

What is DNA polymerase used for?

Uses DNA polymerase to copy single stranded DNA

How does this method make use of ‘dideoxy’ nucleotides which interrupt theability of DNA polymerase to copy DNA?

• DNA polymerase cannot continue to extend the chain ofnucleotides after incorporation of a dideoxynucleotide


• Method known as the dideoxynucleotide chain terminationsequencing


• This method can be automated and allows rapidsequencing of large amounts of DNA

What is DNA normally replicated using?

DNA is normally replicated using deoxynucleotides (dNTPs)

How does DNA polymerase incorporate nucleotides?

DNA polymeraseincorporates nucleotidesby formation ofphosphodiester bondbetween 5’ PO4 group ofnucleotide beingincorporated and 3’ OHon previous nucleotide

DNA polymeraseincorporates nucleotidesby formation ofphosphodiester bondbetween 5’ PO4 group ofnucleotide beingincorporated and 3’ OHon previous nucleotide

DNA sequencing use instead of deoxynucleotides?

DNA sequencing usesdideoxynucleotides(ddNTPs) which do nothave a –OH on the 3’carbon

Why can DNA polymerase no longer incorporate any further nucleotides after incorporation of a ddNTP?

DNA polymerase cannotincorporate any furthernucleotides afterincorporation of a ddNTPas no 3’ OH to formphosphodiester bond

What does the length of fragment depend on in dideoxynucleotide sequencing?

When DNA is replicated by DNA polymerase in presence of dCTP and small amounts of ddCTP some fragments end prematurely. The length of the fragments depends on when ddCTP is incorporated.

To sequence DNA, how many parallel sequencingreactions are done?

To sequence a DNA fragment four parallel sequencingreactions are done, one each for A, C, G and T.


Each reaction contains many copies of the DNAfragment to be sequenced

What happens to the double stranded DNA first?

The double stranded DNA is separated into singlestrands together with a short primer which binds to oneend of the sequence

What happens after the DNA has been separated?

All dNTPs plus a small amount of either ddATP orddCTP or ddGTP or ddTTP are added together with DNApolymerase and a radioactive tracer, 35S

What is dideoxynucleotide (ddNTP)?

deoxynucleotide which do nothave a –OH on the 3’carbon. dNTP is just deoxynucleotides.

What happends in each reaction?

In each reaction the DNA fragment is copied but thereaction will stop at different points where a ddNTP isincorporated.

What happens to the products from each sequencing reaction?

The products from eachsequencing reaction arerun in separate lanes on apolyacrylamide gel.

How does the polyacrylamide gel allow separation of DNA?

The polyacrylamide gelallows separation of DNAfragments differing inlength by a singlenucleotide

How can the position of each fragment be visualised?

Because each DNA
fragment is radioactively
labelled the position of
each fragment can be
visualized by exposing
the gel to X ray film

Because each DNAfragment is radioactivelylabelled the position ofeach fragment can bevisualized by exposingthe gel to X ray film



What do the different bands on the gel represent and how are they read?

The shortest bands are theDNA products closest to theprimer and these travelfastest on the gel; the gel isread from the bottom up, allfour lanes together

What is automated DNA sequencing?

Automated DNA sequencersuse a single reaction for eachDNA sequence, in which allfour ddNTPs are included

How do we indentify which ddNTP was added?

Each ddNTP is labeled with aunique fluorescent marker and we can identify which ddNTPwas added at the end of eachfragment by identifying itsfluorescent colour.

What gel are the DNA fragments separated on?

The DNA sequencefragments are separated on acapillary gel

What are the steps involved in automated DNA sequencing?

•The fluorescent label on eachddNTP has a different wavelength


• As the fragments move down thegel it passes a laser


• The laser light excites thefluorescent tag on each fragmentas it passes


• The wavelength of thefluorescence is read as thefragment passes, and theinformation is recorded

What does the fluorenscence pattern produced show?

The fluorescence patternproduced shows the sequence ofthe DNA

What happens in Large scale sequencing?

• Use restriction enzymes toproduce map of DNAregion.


• Fragment DNA into smallpieces.


• Clone into vector.


• Sequence DNA clonesusing primer in vector.


• Use mapping data andsequence overlap betweenclones to align sequencefrom fragments to getcomplete sequence.

What is next generation sequencing?

Next generation sequencing (NGS) uses sequentialaddition of nucleotides on microchips, short reads of 50-200bp but very rapid, can sequence thousands tomillions of DNA fragments per day to generate over 200-600Mbp per day.

How many protein coding genes are there?

We have sequence information for all genes in genome,current estimate about 30,000 protein coding genes

How much of the genome does protein coding DNA constitute?

This protein coding DNA estimated to be only 2% ofgenome

What is the rest of the DNA?

The 98% non protein coding DNA includes informationfor gene regulatory regions, RNA molecules (tRNA,rRNA etc), introns, untranslated regions of mRNA,repetitive DNA

What has the original sequencing of the human genome allowed us to do?

•investigate basis ofvariation in humans and help to explain individualdifferences in susceptibility to disease, response todrugs or reaction to environmental factors.


• Identify single base pair differences (SNPs (smallnucleotide polymorphisms)) between individuals.


• Can study human evolution and map diseases byinvestigating individuals that share or differ in their SNPs

What are the main model organisms that have been sequenced?

Main model organisms sequenced, E.coli,Saccharomyces cerevisiae, C.elegans, mouse

Why has bioinformatics developed?

Need to analyse large amount of sequence informationhas lead to the development of Bioinformatics

What techniques does bioinformatics use?

Bioinformatics uses computational techniques toorganize, share and analyze this information


• Bioinformatics has required development of interactivedatabases to store and retrieve information.


• Bioinformatics uses mathematical and statisticalapproaches to study the genomic sequences

What else has bioinformatics been developed for?

Bioinformatic approaches have also been developed tostudy dynamics of gene expression

What is structural and functional genomics?

o Identifying features in the genome


o Characterise gene structure, location of introns etc


o Predict gene regulatory regions, promotors,enhancers


o Predicting protein coding regions, protein domains


o Predict gene functions(over 40% human genes stillwith unknown function)


o Identify gene clusterso Identify gene families

What is comparative genomics?

Comparative genomics compares genomesequences between species

What are the purposes of comparative genomics?

• Identify sequences that are conserved overevolution


• Establish phylogenetic relationshipsbetween species


• Identified conserved chromosomal regions– synteny


• Study evolution of genes, gene duplication,exon shuffling, gene fusion, gene density


• Examine relationships between genomesand organisms’ environment

What is the polymerase chain reaction?

Polymerase Chain Reaction (PCR) is powerful technique thathas been recently developed to allow selective amplification ofspecific regions of DNA

What prior knowledge is required for the PCR?

Requires knowledge of sequence information to designprimers that recognise the sequence at the ends of the DNAfragment.

What does Taq do in the PCR?

Makes use of a thermostable DNA polymerase,Taq, toreplicate the DNA region between the two primers

Where does Taq come from?

Taq isolated from bacteria that colonise hot thermal springs

How is PCR an alternative method?

PCR is an alternative method to make many copies(clones) of specific DNA fragments without need to usecloning vectors or restriction enzymes.

How sensitive is PCR?

Very sensitive, able to amplify DNA fragments from verysmall amounts of DNA, 1picogramme (10-12g) of DNA issufficient, but possible to use as little as 1femtogramme(10-15g).

What are some applications of PCR?

Many applications, e.g. in forensics and diagnostics.


Identify contamination in food, e.g. horse DNA in meatproducts

What is the overall mechanism of the PCR?

The PCR uses repeated cycles of targeted DNAreplication to amplify large amounts of a specific DNAfragment.

What are the 3 steps?

PCR involves three steps - denaturation, primerannealing, and extension. These are repeated over and over using a thermocyclerto amplify the DNA exponentially

By how much does the amount of DNA increase with each cycle?

The number of copies of the fragment is doubled in eachcycle, 2,4,8,16, 32, 64, 128, 256 etc. After 30 cycles 230 copies are generated, approx 109copies

What are the templates used for the PCR?

The new strands along with the old strand serve astemplates in the next cycle.

What happens in the denaturation step?

the reaction is heated to  95°C todenature the DNA into single strands

What happens in the primer annealing step?

the reaction temperature isreduced to  45-68°C to allow primers to hybridizeto their complementary sequences in the targetDNA

What happens in the primer extension step?

the reactions temperature israised to 72°C to allow Taq polymerase tosynthesize DNA

What are PCR reactions performed in?

PCR reactions performed in Thermocycler.

How are the Amplified DNA fragments separated?

Amplified DNA fragments are separated by gelelectrophoresis

What is the PCR product size equal to?

PCR product size equal to the amount of DNA betweenthe primers.

What is a limitation of PCR?

A limitation of PCR is that some information about thenucleotide sequence of the target DNA must be known inorder to synthesize primers.

What contamination can occur?

Minor contamination of sample DNA from other sourcescan cause problems

Can PCR amplify long segments of DNA?

PCR cannot amplify long segments of DNA, only thosethat are relatively short.

What are some uses of PCR?

• Can amplify whole genes or parts of genes fromgenomic DNA or cDNA for use in cloning and molecularanalysis.


• Can amplify specific regions of DNA from variety ofsamples, e.g. different individuals.


• 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

What are some applications of PCR?

• PCR is routinely used in screening of mutations involvedin genetic disorders.


• PCR is a key diagnostic methodology for the detection ofbacteria and viruses in humans and pathogenic bacteria,e.g. E. coli, in contaminated food.


• PCR techniques are used to analyse samples fromsingle cells.


• PCR used in forensics to identify individuals from bodyfluids left at a crime scene or in paternity testing.


• Used to check and confirm DNA constructs or transgenicanimals.


• PCR has been used to enforce the worldwide ban onillegal sale of certain animal products e.g. Whale meat

What is Reverse transcription PCR (RT-PCR) used for?

Reverse transcription PCR (RT-PCR) is used to studygene expression by examining mRNA production by cellsor tissues.

What is Quantitative real-time PCR (qPCR) used for?

Quantitative real-time PCR (qPCR) or real-time PCRallows researchers to quantify amplification reactions asthey occur in real time to identify amount of DNA in asample.

What is nucleic acid hybridization?

Method to allow identification of DNA fragmentsor clones containing specific DNA sequences.

What does nucleic acid hybridization rely on?

Relies on complementarity of strands of nucleicacid.

What do the complementary strands do?

Complementary strands of nucleic acid annealto one another.

What can nucleic acid hybridization also do?

Can also be used to identify RNA fragmentscontaining specific sequences.

How are the individual strands of DNA molecules separated?

The individual strands ofDNA molecules can beseparated by heating or inalkali conditions

What happens after the strands are separated?

• After cooling orneutralisation thecomplementary strandswill anneal to one another.


• The complementarystrands “hybridize” to oneanother.

What else does the single stranded DNA hybridize to and what does this mean can happen?

•Single strands of DNA willalso hybridise tocomplementary RNAmolecules.


• Therefore can use ssDNAto identify complementaryfragments of RNA or DNA

What can nucleic acid hybridization be used to do?

• Nucleic acid hybridizationcan be used to identify DNAor RNA that match a specificsequence.


• Can use ssDNA as a ‘probe’to identify specific DNAfragments or clones in acollection that have asequence complementary tothe probe DNA

How is the DNA probe prepared?

DNA must be labelled before it can be used as aprobe.

Why is the DNA labelled?

This allows subsequent detection of the DNA probe

How is the DNA probe labelled?

The DNA probe is labelled by using DNApolymerase to incorporate labelled dNTPs

How are the dNTPs labelled?

dNTPs can be labelled by making them radioactiveusing 32P or by attaching a fluorescent molecule orattaching digoxygenin (steroid molecule)

What is the DNA used to make the probe known as?

The DNA used to make the probeis known as the template DNA.

What then happens to the template DNA?

The template DNA is denaturedand short 6bp primers annealed

What does a Klenow fragment do?

A modified DNA polymerase(Klenow fragment) makes a copyof the template and incorporatesthe labelled dNTPs

What is the resulting DNA probe?

The probe DNA is the labelledDNA fragments copied from thetemplate DNA.

How is the single stranded probe DNA formed?

Single stranded probe DNA isformed by denaturing the labelledDNA

What is the southern blot?

Technique that allows identification of which DNAfragment contains a specific geneor sequence after genomic DNA hasbeen digested with a particularrestriction enzyme. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization

What is the first step of southern blotting?

DNA fragments are separated by gel electrophoresisthen denatured by soaking the gel in alkali

What is the next step?

The DNA fragments are transferred onto a DNA bindingmembrane (nitrocellulose or nylon).

What is then added to the hybridization solution?

Single stranded labelled DNA probe made from specificsequence of interest is placed in hybridization solutiontogether with the membrane with the DNA fragmentsbound to it.

What hybridizes to the labelled probe DNA?

The labelled probe DNA will hybridize to the matchingcomplementary DNA fragment on the DNA membrane.

How is the position of the complementary DNA fragment determined?

The position of the complementary DNA fragment can beidentified as it is labelled by the probe.

What then happens to the resulting restriction enzyme fragment?

This restriction fragment cansubsequently be cloned into a plasmid for analysis and manipulation.

What does a DNA clone southern blot allow?

A clone blot allowsidentification of therestriction fragmentsin cloned DNA thatcontain DNAsequence matchingthe probe DNA.

Is northern hybridization similar to southern hydridization?

Yes, Northern hybridization is a technique related to theSouthern hybridization

What does northern hybridization allow that is different to southern hybridization?

Northern hybridization allows identification of RNAmolecules containing specific sequences

What is the usual use of northern hybridization?

Most often used to identify if a specific gene isexpressed, i.e. transcribed into mRNA, in a particulartissue or sample

Why are some RNA molecules not identified or identified in large quantities?

• Not all genes are expressed at all times


• Many genes are only transcribed in certain tissues or atcertain times.


• Some genes can be transcribed to produce multiplemRNAs of differing sizes

What is mRNA extracted from tissue or sample separated on?

mRNA is extracted from tissue or sample and separatedby size on an agarose gel

Does mRNA need to be denatured before it is transferred to the membrane?

No, mRNA is transferred directly to membrane (mRNA doesnot have to be denatured as it is single stranded)

What is hybridized to RNA on the membrane?

A labelled DNA probe from specific gene is hybridized toRNA on membrane

What is northern hybridization used for?

Used to determine whether a gene is expressed in aspecific tissue by detecting whether a mRNA transcriptcomplementary to the probe is present in the RNAextracted from different tissues.

How is northern hybridization used to look at expression levels of genes in diseases?

Northern hybridization used to determine whether expression levels of a geneis affected by disease by looking for presence andabundance of mRNA transcripts in normal or diseasedtissues.

What else can the northern hybridization blot be used?

• Can determine whetherexpression levels of agene is regulated overtime, e.g. duringdevelopment.


• Can determine size ofmRNA transcript andwhether different sizesof mRNA transcripts aremade at different timesor in different tissues.

What does in situ hybridization involve?

in situ hybridization involves hybridizing a probedirectly to RNA without blotting

Is in situ hybridization confined to tissue sections?

No, in situ hybridization can be carried out in tissue sectionsor entire organisms.

Where is the probe hybridized to the mRNA transcript?

Probe is hybridized to the mRNA transcript in situ, i.e. inthe place where the transcript is made

In what field is in situ hybridization especially helpful?

in situ hybridization is especially helpful in developmentalgenetics to identify where and when genes aretranscribed in embryos.

What else can in situ hybridization identify?

Can identify the cellular and tissue distribution of mRNAsfrom specific genes

What probes are used?

Digoxygenin or fluorescently labelled probes are used

What is an example of in situ hybridization?

• in situ hybridization of myogenin in mouse embryo


• myogenin required for muscle development.


• digoxygenin labelled probe visualised using an antibodywhich recognises dioxygenin and subsequentcolourimetric reaction

How do we use probes to screen cDNAlibraries?

Use nucleic acid hybridisation to identify clones in acDNA library containing specific DNA sequences

What is a cDNA library?

A cDNA library is a collection of plasmids eachcontaining a single cDNA derived from mRNA collectedfrom a specific tissue.

What identification does this screening allow?

Allows identification of cDNA clones for specific gene

What does this identification allow us to grow?

Grow colonies of bacteria, each derived from singlebacteria which contain an individual plasmid clone fromcDNA library

What happens in colony blot hybridisation?

• Bacterial colonies withplasmids containingindividual cDNA clonesare grown on agarplate.


• Colonies aretransferred onto DNAbinding membrane.


• Bacteria are lysed andDNA denatured usingalkali.


• Membrane hybridisedwith labelled DNAprobe

What is the membrane with radioactive probe bound exposed to?

Membrane with radioactive probe bound is exposed to Xrayfilm.

Which bacterial colonies do you identify?

Allows identification of the bacterial colony on the platecontaining specific cDNA clone

How do you isolate plasmid DNA with cDNA clone?

Grow up single colony to isolate plasmid DNA with cDNAclone for subsequent analysis

What are DNA microarrays?

DNA microarrays or gene chips are modern deviceswhich use nucleic acid hybridisation to rapidly measurewhich genes are expressed in a tissue sample

What do DNA microarrays enable to happen?

Enables the ability to simultaneously identify all thegenes expressed in a particular sample.

what approach do DNA microarrays take?

Uses a reverse approach whereby all the mRNA from asample is converted to cDNA, fluorescently labelled andused as probe.

What is this cDNA probe applied to?

This probe is applied to the array which contains spotswhere single stranded DNA from each gene in thegenome is laid out in an array.

What is spotted on the microarray slide?

ssDNA (oligonucleotides) matching all genes in genomeare spotted onto microarray slide

Draw a diagram of DNA microarray technique

What does the labelled cDNA bind to on the slides?

the labelled cDNA will bind to the spots on the slides thatcontain complementary sequences

How are the genes expressed in the sample identified and their level of expression measured?

• the spots that bind the labelled will then fluoresce underlaser light in a piece of equipment known as a chip reader


• the location and brightness of the fluorescent spots on themicroarray identify the genes expressed in the test sampleand their level of expression

How is the relative expression level determined?

Labelled spots on microarrayidentify expressed genes.Relative fluorescent intensityindicates relative expression level

What do microarrays enable the study of?

Microarrays uses nucleic acid hybridization to enable thestudy of gene expression from a genomic perspective,i.e. study of the expression dynamics of all genes in thegenome.

What is the transcriptome?

The set of transcripts present in a cell, tissue or sampleis called the transcriptome

What is the study of transcriptomes known as?

• The study of transcriptomes is known as transcriptomics.This level of genetic study is becoming a powerfulmethodology in modern molecular genetics.


• Allows the study of which genes are expressed underany type of conditions or situations

What is comparative microarrays?

• DNA microarrayscan be used todirectly comparegene expressionin two samples


• Can be used tocompare geneexpression innormal anddiseased cells