Molecular Genetics Technologies Flashcards

Question

How is bacteriophage lambda converted to a cloning vector?

Answer 1

Normal genome can't accommodate large inserts as size constraint of fitting into phage head Can remove section of DNA into involved in lysogenic Allows inserts up to 10kbp

Answer 2

Non-essential region can be deleted & substituted with 'stuffer' region containing restriction enzyme sites to create replacement vector Stuffer region ensures genome large enough to be packaged into phage Stuffer region can be removed by restriction enzyme digestion & ligated to new DNA up to 23kbp

Answer 3

Similar to plasmid Have a lambda cos site Ligated with fragments of DNA ~35kbp in length High conc. ligation produces catenane Phage particles containing cosmid & DNA insert efficiently infected into cells On entry cosmid and insert circularises

Answer 4

Construct genome/cDNA library Screen for desired clone OR Use PCR to directly amplify up specific sequences from genomic DNA/cDNA made by reverse transcription of mRNA

Answer 5

Constructed from cloning overlapping DNA fragments - generated by partial digestion of genomic DNA with restriction enzyme Multiple segment endpoints within the population of fragments - inserts overlap Partial digest size needs to be compatible with vector insert size capacity

Answer 6

Lambda DNA central region of EMBL3 vector called shutter fragment replaced by insert produced by partial digestion of genome of interest Plating high titre of infectious phage particles mixed with E. coli Mixed with soft agar and plated, 1 plaque represents 1 ligation event Enough plaques to represent entire genome Can make a filter lift & hybridise with probe OR pick plaques & PCR

Answer 7

Several clones are detected by probe and can be aligned Terminal regions of these clones can be used to ID new probes to screen library to ID further clones with overlapping inserts Overlapping clones constitute 'contig'

Answer 8

BACs = engineered derivatives of E.coli F factors Insert sizes 250–300 kbp, electroporated into cells Clones robotically picked into 96 well plates DNAs prepared and IDing specific clone done by combinatorial PCR screening using gene specific primers

Answer 9

cDNA prepared by reverse transcription of mRNA Libraries representative of the RNA population Methylating cDNA with EcoRI methylase Ligate EcoRI linkers to ends Cut with EcoRI producing cDNA with EcoRI cohesive ends cDNA fragments into lambda insertion vectors

Answer 10

By hybridisation: DNA/RNA’ oligonucleotide probes By PCR Using antibodies to expressed proteins Functional cDNA screens in appropriate cell types

Answer 11

Need some knowledge of sequence to design primers – can use a pool of degenerate oligonucleotide primers Target DNA can be genomic DNA/cDNA PCR product cloned directly into plasmid vectors TOPO cloning uses vector with T overhang & topoisomerase linked to ends; combine with Taq PCR product that has an extra A on each end – very efficient

Answer 12

Plasmids vectors with DNA sequences from multiple sources for many applications Reporter expression vectors with fluorescent markers, used to ID cells Shuttle vectors with separate replication origins, selection markers and promoters

Answer 13

Introduce single nucleotide changes, insertions, or deletions using synthesised oligonucleotides Change AA sequence of protein Create/remove restriction site Alter gene regulating regions to ID key sequence

Answer 14

Parental strands methylated at DpnI sites Denature parent strands Anneal oligonucleotide primers and add dNTPs and Pfu polymerase DpnI enzyme added and only cuts methylated DNA Even if only one strand cut the strands are destroyed only leaving both strands mutagenised

Answer 15

Golden Gate Assembly - utilises type IIS restriction endonucleases to create unique cohesive ends, no restriction sites remaining Gibson Assembly - allows DNA molecules to be joined via annealing short end regions of complementarity, no sequence 'scar', few constraints on application

Answer 16

Type IIS restriction enzyme eg. BsaI, cleaves away from recognition site at diff positions on each strand, results in staggered ends Positioning of BsaI recognition sites flanking seqs of interest allows DNAs cut to create compatible ends for ligation Results in seamless ligation product with no BsaI sites left on starting DNAs Digestion and ligation can occur simultaneously

Answer 17

Sequence independent seamless joining of DNA molecules, isothermal reaction Require to link regions A and B from separate plasmids and insert into vector PCR primers each contain 3’ region of complementarity to plasmid template and 5’ tail with left or right end sequence of other plasmid 5’ ends of PCR products degraded by T5 exonuclease, leaves 3’ single strand regions Annealing between single strand regions at 50 C Polymerase - DNA synthesis from 3’ ends to fill gaps Polymerase catches up exonuclease & nicks sealed by ligase, covalently closed circular molecule

Answer 18

Sanger method Maxam Gilbert method Both depend on fractionation of ssDNA in denaturing polyacrylamide gels to give 1 nucleotide size difference resolution

Answer 19

1 dNTP labelled Ratio dNTP & ddNTP in each reaction determines probability of chain termination at each position: uses template strand and incorporates dNTPs (high probability) or ddNTPs (low probability), 1 min for each base and run on gel

Answer 20

Can do all reactions at once in one gel as 4 fluorescent dyes used Can be run through capillaries, automated, use T7 polymerase Limit of 500-1000 good sequence

Answer 21

Use genome molecularly cloned in BAC library, overlapping clones ID'd Each BAC cut into many smaller random fragments & 'shotgun' cloned into plasmid vectors Cloned DNA in plasmids sequenced using universal primer & final sequence assembled from sequence overlaps

Answer 22

All rely on methods that sequence DNA directly Genomic DNA reduced to small random DNA fragments by sonication - represent all sequences of starting DNA DNA fragment library produced and modified in various ways by adapter ligations and by PCR Rely on massively parallel sequencing in single procedure Each reaction occurs on discrete solid phase clonal amplification of single DNA molecule - polony Simultaneously monitored by detector system

Answer 23

Sequencing by synthesis w/ DNA polymerase & fluorescent terminators Short ss oligonucleotides of 2 defined sequence (P5 & P7) chemically bonded via 5' with 3' ends free to glass slides to form a dense lawn Adapters w/ same sequence as bonded oligonucleotides ligated to each end of sonicated DNA - ligated so all have different adaptor DNA population denatured & added, attaches via complimentary base pairing to bonded oligonucleotides - primers DNA pol & dNTPs added & DNA molecule copied from 3' end by P5 & P7 Original DNA washed away Copy under renaturation conditions to adjacent P5, next round DNA synthesis dNTPs added for sequencing w/ diff fluorescent groups & reversible termination block

Answer 24

``` Whole genome re-sequencing Gene expression ChIP sequencing microRNA discovery Targeted re-sequencing ```

Answer 25

DNase I hypersensitivity: map open regions of chromatin structure, open = regulatory regions like enhancers/promoters, open regions cut by DNase I, detected by probe ChIP: detects sequences associated w/ specific chromatin modifications/binding of specific transcription factors, immunoprecipitated DNA detected by region-specific PCR primers

Answer 26

Can generate huge no. sequence reads & can be used as sequence 'counters' To understand content of DNA extracted a short read/pair of reads of sequence generated from each original molecule obtained from chromatin sampling 25 – 35 bp sequence allows informatics methods to map location to reference genome