Gene cloning

Question 1

what is gene cloning?

Answer 1

• manipulation of DNA in a test tube
• returning modified DNA to study function of gene products
• isolation of defined pieces of DNA from the genome
• used to study genetic disease to bioengineering of pharmaceutical products

Question 2

DNA extraction

Answer 2

• DNA can be extracted from any nucleated cell
• extraction method must not degrade the DNA
• enzymatic hydrolysis
• mechanical sheering

Question 3

how and why do we use purified enzymes to manioulate DNA?

Answer 3

• These manipulations, carried out in vitro, provide the foundations for gene cloning anf stdying DNA in biochemistry, gene structure and control of gene expression
• Within their original cells these enzymes participate in essential processes such as DNA replication, transcription etc.
• After purification the enzymes can be persuaded to carry out their natural reaction under artificial conditons

Question 4

What is a recombinant DNA molecule and how is DNA manipulated?

Answer 4

• A holy grail of molecular biology
• constructed by combining 2 or more fragments of DNA (vector and gene of interest)
• the vector and DNA must be cut at specific points and joined together in a controlled manner
• Cutting and Joining DNA molecule are 2 famous examples of DNA manipulation
• DNA can also be shortened, lengthened, copied into RNA or new DNA, and modified by the addition or removal of specific chemical groups

Question 5

Nucleases ?

Answer 5

• Enzymes that cut, shorten or degrade nucleic acid molecules
• breaks the phosphodiester bonds that link 1 nucleotide to the next in a DNA strand
• 2 kinds:
  1. Exonucleases: removed nucleotide one at a time from the end of a DNA molecule (eg. Nuclease BAL31, E. coli Exonuclease lll)
  2. Endonucleases: break internal phosphodiester bonds within a DNA molecule (eg. S1 Nuclease, Mung Bean Nuclease DNAsel, Restriction enzyme)

Question 6

RNAses?

Answer 6

• RNAseA: endoribonuclease that specifically degrades single stranded (ss) RNA
• RNAseH: Endoribonuclease that digest the RNA of an RNA-DNA hybrid

Question 7

Ligases?

Answer 7

• covelently links the free ends of DNA molecules
• repair single stranded (ss)-break in one of the strands of a double stranded (ds) molecule
• it also joins together individual DNA molceule or the cohesive ends of the same molecule
• mode of action; catalyses the formation of a phosphodiester bond between adjacent 3’-OH and 5’-P termini in DNA
• ligation of complementary sticky ends is much more efficient than two blunt ends
• Hydrogen bonding gives a relatively stable structure for the enzyme to work on

Question 8

Polymerases?

Answer 8

• synthesise a new strand of DNA complementary to an existing DNA or RNA template
• most polymerases need a template
• 4 types used routinely in molecular biology techniques;

DNA Polymerase 1, Klenow fragments DNA Polymerase, Reverse Transcriptase, TaqDNA Polymerase

Question 9

1. DNA Polymerase 1?

Answer 9

• Usually from E. coli and T4 phage
• DNA dependant DNA polymerase
• 5’ to 3’ polymerase, 5’ to 3’ exonuclease and 3’ to 5’ exonuclease
• commonly used in Nick translation and Probe preparation, repairing of DNA fragments, producing blunt ends from sticky ended DNA

Question 10

2. Klenow fragment DNA polymerase ?

Answer 10

• DNA dependant DNA polymerase
• Having 5’ to 3’ Polymerase & 3’ to 5’ exonuclease activities
• No 5’ to 3’exonucleaseactivity
• Can only synthesis a complementary DNA strand on a single stranded template
• Commonly use in Sanger dideoxy sequencing, synthesis of second strand cDNA in cDNA cloning, Filling in the 3’ recessed termini created by digestion of DNA with RE & labelling the termini of DNA fragment -end filling reaction.

Question 11

3. Reverse transcriptase?

Answer 11

• needs RNA as a template
• having 5’ to 3’ polymerase, 5’ to 3’ tiboexonuclease and 3’ to 5’ exoribonuclease activities
• commonly used in the synthesis of cDNA for cloning
• labelling the termini of DNA fragments with protruding 5’ ends

Question 12

4. TaqDNA Polymerase?

Answer 12

• 5’ to 3’ polymerase activity only (no proofreading activity)
• used in PCR (requires specific primers)
• high polymerase activity , copies ~ 1 kb/min
• latest version of Taq has proofreading actitivities with even higher polymerisation capabilities

Question 13

DNA modifying enzymes?

Answer 13

• modify DNA molecules by addition or removal of specific chemical groups
  1. Alkaline phosphatas (AP); removes the Phosphate froup present at the 5’ terminus of a DNA molecule - prevents recircularisation of plasmid during cloning
  2. Terminal deoxynucleotidyl transferase; add 1 or more deoxynucleotides onto 3’ terminus of a DNA molecule
  3. DNA Methylase (dam and dcm); transfer of methyl group to internal A or C residues in the specific sequences to produce methylated duplex DNA - protection of DNA from restriction enzymes
  4. Polynucleotide Kinase; Adding phosphate groups on to free 5’ termini (reverse of AP)

Question 14

the three classes of endonucleases?

Answer 14

I - recognises specific sequences, but not particularly useful in gene manipulation as their cleavage site is non-specific, they have methylase activity

II - they are Mg2+ dependant with a highly specific recognition site. very useful for DNA manipulation

III - contain nuclease and methylase activity. recognition site is not symmetrical

Question 15

what are restriction enzymes?

Answer 15

• class II endonucleases
• enzymes for cutting DNA in a precise and reproducable manner during molecular cloning work
• cut both strands of DS DNA within a (normally palindromic) recognition sequence
• hydrolyse sugar phosphate backbone to give 5’ phosphate on 1 side and 3’ -OH on the other
• yield blunt or sticky ends
• discovery of them = breakthrough in development of genetic engineering

Question 16

Restriction endonuclease nomenclature?

Answer 16

• Smith and Nathans (1973)
  1. species name of organism that produces the enzyme eg) Eco
  2. strain will be written after species eg) EcoR (if more than one roman numerals added eg. EcoRI)

Question 17

Isoschizomers?

Answer 17

• restriction endonuclease that recognise the sample sequence.
• the first example discovered is called a prototype, all subsequent enzymes that recognise the same sequence are isoschizomers

Question 18

Neoschizomers?

Answer 18

• recognise the same sequence, but cleave at different positions from the prototype

Question 19

gel electrophoresis?

Answer 19

• separate DNA fragments of different sizes
• need a chemical dye to see the DNA (ethidium bromide, SYBR green or gold) either fluorescent dye (poor resolution but quick and easy) or radiactive labelling followed by autoradiography (dangerous)
  1. place mixture of DNA restriction fragments in the well of an agarose or polyacrylamide gel
  2. apply electric field
  3. molecules move through pores in gel at rate inversely proportional to their chain length

Question 20

Pulse Field Gel Electrophoresis?

Answer 20

• separation of large DNA molecules by changing periodically the electric field from a gel matrix
• in a standard gel; DNA molecules bigger than 15Kb move togehter regardless of their size
• PFGE is a variation that induces alternating voltage gradient to improve the resolution of larger molecules
• the voltage is periodically switched among 3 directions; 1 - to the central axis of the gel, 2, 3 - at an andle of 60 degrees from both sides
• results in DNA thats not moving in a straight line through the gel but in a ‘net forward’ migration pattern

Question 21

Traditional approach to identifying the presence of genes within the genome?

Answer 21

• hybridisation with radioactively labeled DNA or RNA probes - complementary to the sequence we are trying to identify
• gives quantitative and qualitative information about the presence of the gene/genes
• this has now been replaced by PCR and QPCR, still needed for ultimate clarification

Question 22

Denaturationg of DNA?

Answer 22

• Double stranded (ds) nucleic acids can lose their secondary structure by denaturation
• its promoted by heat, extreme pH, hydrogen bond breaking agents (eg. concentrated Urea)
• denaturation involved breaking the hydrogen bonds in base pairs and separating the strands of the double helix
• the temperature at which a (long) DNA double strand denatures depends on its base pair composition - higher G+C content = higher temp (held together with 3 hydrogen bonds, A-T = 2)

Question 23

DNA hybridisation?

Answer 23

• the process by which ds DNA/RNA strands reform a double helix
• separated strands will rejoin if transferred to conditions favouring base pairing (lowering temperature, higher salt conc., removing denaturants)
• hybridisation can only occur complementary DNA strands (or almost) - in a mixture of single strands each strand seeks out its original partner.
• process can be long (days) if DNA is dilute or contains many sequences

Question 24

the membrane-hybridisation assay?

Answer 24

• use hybridisation of complementary regions to identify the presence of a gene in a population
  1. melt ds DNA to form ss DNA
  2. DNA binds to filter (bind and dont move)
  3. strands incubated with labelled DNA
  4. complementary DNA hybridises
  5. wash away labelled DNA that did not hybridise to DNA bound to filter
  6. perform autoradiography
• tells us that the DNA of interest is present in our sample

Question 25

southern blotting?

Answer 25

• detects specific short DNA fragments/gene of interest in a mixture of DNA
• used for applications like determining how many genes corresponding to a particular cDNA probe are present in an organisms genome
• many proteins are encoded by small gene families rather than a single gene
  1. DNA cleaved with restriction enzymes
  2. gel electrophoresis separares DNA fragments
  3. transfered to nitrocellulose membrane via capillary action from the gel in alkaline solution (denatures the DNA - releases the single strands across filter paper)
  4. hybridised with labelled (complementary to the gene of interest) DNA probe in autoradiogram
  5. only the pieces of DNA that hybridised with the probe will show up when exposed to x ray
• similar technique is Northern blotting where RNA is separated by gel electrophoresis and transferred to a filter.

Question 26

northern blotting?

Answer 26

• standard method of analysing mRNAas the target
• gives size and amount
  1. separate RNA by agarose gel electrophoresis by size
  2. blot RNA onto nylon or nitrocellulose membrane
  3. incubate membrane with labelled probe
  4. locate hybridising bands by autoradiobiography
• dark bands appear where our probe binds to the mRNA - we can see size of it

Question 27

Polymerase Chain Reaction?

(what is it, what does it require)

Answer 27

• Kary Mullis 1984
• molecular photocopier - picks out parts to amplify
• DNA sequences can be amplified by PCR for use in cloning, as probes and in forensics (generally low amount of DNA)
• PCR can be used to amplify rare specific DNA sequences from complex mixtures (when the ends of the sequences are known)
• PCR of mutant alleles allows for detection of human genetic diseases
• requirements for the reaction:
  1. Taq DNA polymerase - copies original and generates new DNA strands
  2. dNTPs - nucleotide bases that form the new DNA strands
  3. Oligonucleotide primers - forms initiation site for Taq polymerase
  4. Mg2+ ions - stabilises the primer: DNA duplex, required for correct dNTP incorporation
  5. a template - to work with

Question 28

steps of PCR?

Answer 28

Cycle 1:

• test tube with DNA, nucleotides, Taq polymerase and lots of primers
  1. Denaturation of DNA at 90^oC - if high G/C content then its 98^oC
  2. annealing of primers at 60^oC
  3. elongation of primers at ~70^oC (complementary base pairing forms ds) (using TaqPolymerase)
• repeat for however many cycles to get a greater proportion of the target gene

Question 29

design of PCR primers?

Answer 29

• primers should be ~20 bases long
• G/C content should be 45-55%
• the annealing temps should be within 1 degree C of one another
• the 3’ - most base shoule be a G or a C
• the primers must not base pair with eachother or themselves (hairpins)
• primers must avoind repetitive DNA regions
• Primer3Plus program at the Whitehead institute is the most reliable and versatile tool to design primers
• we can use the PCR primers to introduce specific sequences of DNA into the product for gene cloning

Question 30

how do you optimise the PCR reaction?

Answer 30

• annealing temperatures of the primers
• conc. of Mg²⁺ in the reaction
• the extension times
• extention temperature
• the amount of template and polymerase - more is less)

Question 31

Optimising the annealing temperature for PCR?

Answer 31

• primers have calculated annealing temperature
• temperature must be confirmed practically
• temperature steps of 2^oC above and below
• use gradient cycler

Question 32

Optimising Mg²⁺ concentration in PCR?

Answer 32

• fidelity of the PCR depends on Mg²⁺
• Mg²⁺ should be varied in steps of 0.5 mM
• sometimes a comprimise between yield and specificity

Question 33

primers that form hairpins?

Answer 33

• a primer may be self-complementary and be able to fold into a hairpin
• the 3’ end of the primer may be base-paired (for example) preventing it annealing to the target DNA

Question 34

primers that form dimers?

Answer 34

• a primer may form a dimer with itself of with another primer
• primer dimers can be an excellent, but unwanted, substrate for the Taq polymerase

Question 35

DNA cloning with vectors?

Answer 35

• recombinant DNA technology depends on the ability to produce large numbers of identical DNA molecules (clones)
• clones are generated by placing a DNA fragment of interest into a vector DNA molecule, which can replicate in a host cell resulting in large numbers of the fragment with the vector
• two common vectors are E. coli plasmid vectors and bacteriophage lamda vectors
• plasmid cloning vectors are extrachromosomal self-replicating DNA molecules

Question 36

Bacterial plasmids as vectors?

Answer 36

• bacterial plasmids are circular ds DNA molecules
• they’re extrachromosomal - separate from the main genomic DNA
• 2-200kb in size
• exist in multiple copies (a few to 100’s) in the bacterial cell
• function in nature is to enhance adapatation to environment of bacteria by transfering genes horizontally
• reason for antibioitic resistant pathogenic bacteria

Question 37

two vital features of plasmids?

Answer 37

1. a replication origin, enabling the plasmid to be replicated independantly of the bacterial genome
2. a gene which confers selective advantage on the organism (ie. antibiotic resistance) and selects for the plasmid

Question 38

replication via bacterial plasmids ?

Answer 38

• plasmids tend to be spontaneously lost when no selective advantage is gained from them
• they’re normally present at low levels
• the replication origin defines plasmid copy number per cell
• plasmids are passed onto daughter cells when fission of bacterial cells occurs
• only one plasmid of a given replication group can be stably maintained in a bacterial cell although more than one copy is usually present
• plasmids from different replication group can be stably maintained in a bacterial cell simultaneously - requires simultaneous selection for different characteristics carried on different plasmids
• plasmid cloning allows isolation of DNA fragments from complex mixtures

Question 39

extracting the plasmid from from bacterial cells?

Answer 39

• plasmids can be purified easily from bacteria after cell lysis
• purification relies on physical properties of plasmid DNA vs. genomic DNA
• result is circular ds DNA molecule - usually added with a supercoil
• yields are low, depending on plasmid copy number per cell
• resulting DNA is suitable for further operations, including restriction with endonucleases, DNA sequencing template etc.
• plasmid purification from small-scale (1-10ml) bacterial cultures is now automated

Question 40

reintroducing plasmids into bacterial cells?

Answer 40

1. by transformation with ‘naked’ circular ds plasmid DNA
2. bacterial cells are made ‘leaky’ by treatment with membrane permeabilising agents (eg. calcium ions) - these cells are called competent cells
3. competent cells are mixed with DNA and uptake can be increased by electroporation = electric shock treatment
   - DNA is taken up at very low efficiency - 1 / 10,000-1mil cells but plasmid then replicates in cell and daughters
   - efficiency of transformation decreases as size of circular DNA increases - >10kb = hard to transform
   - cells which have taken up the plasmid DNA can be isolated if a suitable selectable gene is present on the plasmid

Question 41

what is blue/white selection?

Answer 41

• E. coli cells are engineered to synthesise an incomplete, inactive enzyme the w-peptide (omega peptide)
• cells containing the original vector plasmid also synthesise a small peptide fragment from B-galactosidase, the a-peptide (alpha peptide)
• this binds to the w-peptide to give active B-galactosidase
• cells with original plasmid produce a blue colony when treated with X-gal
• in plasmids with an insert, the gene is interrupted, the a-peptide is mutated and colonies are white

Question 42

Bacteriophage lambda and gene cloning?

Answer 42

1. the middle of the lambda chromosome contains dispensible genes, the central stuffer, which are removed with a restriction enzyme
2. DNA to be cloned is cut with the same restriction enzyme and ligated in place of the central gene cluster in betweeen the arms
   - Vectors designed for cloning genomic DNA accepts fragments of 12-20 kbp
   - Vectors designed for cloning cDNA accept fragments in the range 0-10kbp
3. the recombinant chromosome is packaged into phage heads to form a recombinant virus
4. virus infects bacterial cells and replicated its DNA
5. the phage lyses the cells and forms plaques on a cell lawn from which the cloned DNA can be recovered
   - either undergos lytic replication or lysogeny following infection of E. coli
   - The lytic cycle: The phage infects a bacterium, hijacks the bacterium to make lots of phages, and then kills the cell by making it explode (lyse), COS sites at the correct spacing (43-53kb) allows spontaneous formation of complete, functional lambda phage
   - The lysogenic cycle: The phage infects a bacterium and integrates its DNA into the bacterial chromosome, allowing the phage DNA (now called a prophage) to be copied and passed on along with the cell’s own DNA (host isnt killed)

Question 43

how does the bacteriophage lambda replicate and package its DNA?

Answer 43

• rolling circle mode of DNA replication
• endonuclease A cleaves at cos sites
• lambda proteins assembled DNA packaged in phage head (upper limit = 53kb)
• produces infective particals

Question 44

comparing plasmid and phage cloning vectors?

Answer 44

PLASMID:

• circular DNA
• DNA to be cloned inserted into vector
• transformation efficiency = good
• transformation falls off rapidly with insert
• better for small inserts (<5 kbp)
• bacterial clones containing plasmid = easy to handle
• plating density of clones = limited (1000 per plate)
• plasmid DNA - easy to prepare
• better for small libraries, subcloning, DNA manipulation and routine purposes

PHAGE: (better choice)

• linear DNA
• DNA to be cloned replaces part of vector
• transfection efficiency = better and not affected by size of insert
• better for larger inserts (>5 kbp)
• phage clones more difficult to handle (lawns)
• plating density of phage = high (>10,000)
• phage DNA
• harder to prepare
• better for large libraries

Question 45

constructing DNA libraries with lambda phage and plasmid cloning vectors?

Answer 45

• cloning all the DNA of organisms into plasmid vectors is not practical due to low transformation efficiency of E. coli and small number of colonies that can be grown
• bacteriophage do not suffer from such limitations
• collection of clones that includes all the DNA sequences of a given species is called a genomic library

- this library can be screened for clones contraining a sequence of interest

Question 46

production of cDNA libraries?

Answer 46

• prepared from isolated mRNAs
• reverse transcriptase makes DNA copy from RNA template, requires primer
• linkers may be added to the ends of DNA to help join it to vector

Question 47

comparing genomic and cDNA libraries?

Answer 47

GENOMIC:

• cloned DNA is directly from genome
• generally only library necessary for prokaryotes
• cloned fragments contain both transcribed and non-transcribed regions / introns + exons
• cloned fragments have no poly(A) tails
• library same when made from DNA of any tissue or developmental stage
• need to contain more clones to represent entire genome (>10⁶)

cDNA:

• cloned DNA fragments are copies of mRNA sequences
• both types of library necessary for eukaryotes
• cloned fragments contrain only transcribed regions / exons
• have poly(A) tails and other mRNA modifications
• libraries made from different tissues or stage differ as dif genes transcribed to mRNA
• need to contain fewer clones to represent all mRNA sequences present (<10⁶)

Question 48

screening a library?

Answer 48

• library = collection of clones
• need to identify the E. coli colonies or plaques that contain the DNA of interest based on detection of:
  1. the gene product - enzyme activity / protein (impractical as gene may not be expressed / inactive
  or. ..
  2. the DNA sequence

Question 49

screening on the basis of activity?

Answer 49

EXAMPLE: cloning amylase gene, amylase degrades starch so;

• plate out library (E. coli) on starch containing agar
• gene containing amylase gene will have halo where starch digestion has taken place

Question 50

screening with antibodies?

Answer 50

• screening on the product of the gene but not the activity (need for when E. coli cant produce a functional enzyme)
• antibodies are used to detect specific protein and amount of protien in a complex mix - recognise a particular antigen within the protein (specific)
• antibodies are produced by B cells (wbc)
• you must have the protein your looking for
  1. purify protein and inject into animal to raise antibodies (bind specifically to it)
  2. library plated out on agar plate
  3. transfer the colonies onto a filter (nitrocellulose or nilon based filter)
  4. lyse the cells to release the protein (nitrocellulose has high affinity for binding proteins)
  5. (antibodies themselves however are proteins so it may bind them too) we must add inert protein to block non-specific binding (e. bovine serum albumin which binds to the entire surface)
  6. add radioactive antibody which binds to specific colony it recognises
  7. expose to X-ray film and darken where the antibody degrades

Question 51

screening on the basis of DNA hybridisation? (screening on the basis of DNA)

Answer 51

• overcomes problems of searching for a probe based on the activity of product
• nucleic acid hybridisation used to detect a specific nucleic acid sequence in a complex mix using a DNA probe (from PCR or from a pre-made cDNA library) (complementary base pairing)
• can take place in solution or with target DNA attached to filter (nylon / nitrocellulose)
• used to screen both genomic and DNA libraries
  1. plate out library of clones on agar plate
  2. transfer to a filter (nitrocellulose / nylon)
  3. lyse cells and denature DNA with alkali (need ss)
  4. add inert salmon sperm DNA to block non-specific binding (nitrocellulose binds DNA as well as proteins)
  5. incubate with radioactive DNA probe (remove the denaturant, the heat/alkali, and wash away any non-bound probe)
  6. expose to x-ray film (darkens the film where the probe has been taken up)

Question 52

looking at differentially expressed genes using screening?

Answer 52

• we can compare libraries of different tissue types to see which genes are specific to certain tissues

EXAMPLE: genes that are expressed in one tissue type but not another type

1. make cDNA library from liver
2. Hybridise with labelled total cDNA from liver (all clones will show up on x-ray film) and muscle (lots of the same spots appear but some absent)
   - clones hybridising only to liver cDNA are differentially expressed
   - if spots appear with both tissue cDNAs theyre common (probs general housekeeping enzymes/general cellular processes that occur in all tissue types) between them and if they dissapeared with the muscle cDNA we can say those products are specific to muscles

Question 53

Sanger sequencing: what is it and discovery?

Answer 53

• characterising a cloned sequence; DNA sequencing - determining the sequence of nucleotides
• sanger dideoxynucleotide chain termination method (automated or manual)
• slow but is gold standard of methods of choice and most common
• NGS is massively parallel - high-throughput sequencing - hundreds to thousands of genes at one time
• Fred Sanger etc. mid 1970’s won 2nd nobel prize in Chemistry in 1980
• based on principle that ss DNA molecules that differ in length by just 1 nucleotide can be separated by polyacrylamide gel electrophoresis (agarose doesnt have resolution to do this) (vertical)

Question 54

sanger sequencing: Dideoxynucleoside triphosphates?

Answer 54

• ddNTP
• derivatives of the normal deoxyribonucleoside triphosphates that lack the 3’ -hydroxyl group
• has an H on the 3’ rather than an OH which doesnt allow the chain to be extended
• if polymerase incorporates one of these the chain will stop growing at that size

Question 55

Sanger sequencing: method?

Answer 55

1. purified DNA synthesised in vitro in a mixture that contains ss molecules of the DNA to be sequenced, DNA polymerase, and the 4 deoxyribonucleoside triphosphates (dNTPs)
2. if a dideoxyribonucleotide analog of one of these is also present it competes with an excess of the normal dNTP, it can be incorporated occasionally, at random, into the growing DNA chain - blocking addition of next nucleotide.
3. the reaction mixture will eventually produce a set of DNAs of different lengths complementary to the template DNA thats being sequenced and terminating at certain base (eg. using ddATP) which shows us where each of the A’s lie.
4. repeat for all 4 bases run in individual reactions –> electrophoresis separated in 4 parallel lanes
5. the newly synthesised fragments are detected by a label (radioactive or fluorescent) that has been incorporated into the primer or onto one of the dNTPs
6. in each lane the bands represent fragments that have terminated at a given nucleotide
7. read off the bands starting at the bottom working across the lanes, the DNA sequence of the newly sequenced strand can be determined

Question 56

sanger sequencing: issues and improvements?

Answer 56

• 15-20 years ago it took far too long to do these reactions
• until recently, sequencing was conducted on polyacrylamide slab gels
• 100 bases could be sequenced each run (24-48hrs)
• turn around not good enough for sequencing genomes
  improvements: made it an automated system:
• fluorescently labeled ddNTPs - allows all 4 termination reactions to be run in 1 lane (detected by laser)
• throughput improvements - one tube reaction, no radioactivity, improved resolution of close bands
• 1mbp every 24 hours now = machines with 96 capillaries tubes filled with separating polymer, DNA travels through the tubes
• polyacrylamide gel - better resolution
• polymerase - better processivity

these have increased speed, accuracy and read length (max is 1000bp however - it gets difficult to look at the small percentage changes in a larger molecule)

Question 57

DNA sequencing of large cloned inserts?

Answer 57

• we need strategies that allow us to sequence large inserts (sanger not great)
• primer walking - requires very fast and accurate analysis of sequence reads since each sequencing reaction uses information from the previous read
• shotgun sequencing - takes max advantage of the speed and low cost of automated sequencing but relies totally on software to assembly a jumble of sequence reads into a coherent and accurate contig

Question 58

Primer walking (sequencing)?

Answer 58

1. large kb of DNA to be sequenced (up to ~4kb)
2. get a primer that is complementary to 5’ end of our strand and sanger sequence it (~800bp)
3. look at 3’ end of the new sequenced strand and design a new primer matching that
4. keep doing it again and get another ~800bp of sequence
5. entire sequence generated by joining the strands that overlap (contiguous pieces of DNA) in silico
   - this way we can cover the whole of the gene of interest
   - we could sequence from both ends and sequence to the middle til overlap
   - problem: slow speed (making primers each time takes time) - use a shotgun for longer strands

Question 59

Shotgun sequencing?

Answer 59

• for larger DNA strands (eg. 20kb)
  1. insert cut into small fragments by restriction enzymes (more than 1 to cut at different positions in the sequence) or shatter the DNA (random) and sequence each quickly
  2. clone gene fragments in a plasmid vector (~1kbp)
  3. sequence insert
  4. remove the vector sequence present in the sequence using same software as below
  5. complex contig assembly using specialist software eg) DNASTAR, BIOEDIT, Sequencher
• both the forward and reverse strand can be sequenced separately using specific primers (both directions)

Question 60

open reading frames?

Answer 60

• genes that encode proteins = ORFs withing the DNA sequence
• ORF starts with start codon (usually ATG)
• ends with stop codon (TAA, TAG, TGA)
• DNA has 6 reading frames
• we expect a stop codon every 100-200 bp by random so if not one seen for a while = ORF
• ORF scanning located genes
• BLAST search look for homologous genes/proteins; how well they align with genes of known function
• Clusters of Orthologous Groups of proteins (COGs)