Genomic & cDNA Libraries Flashcards
What is the difference between a genomic and cDNA library?
Genomic libraries include clones of the whole genome, cDNA only includes clones that correspond to mRNA sequences (no introns)
Describe the process of oligo-dT chromatography
A form of chromatography with an oligo dT matrix (poly T stretch attached) onto which mRNA (with it’s poly A tail) may bind. Only binds mRNA as other forms of RNA do not have the poly A tail. mRNA eluted by high salt conc. to break A-T bonds
What are the steps involved in creating cDNA from mRNA (5)?
- Solution creation: Start with mRNA, add reverse transcriptase, dNTP and oligo dT primer
- dNTP addition: Primer binds to poly A tail of mRNA, reverse transcriptase adds dNTPs from 5’ to 3’
- RNA removed: Ribonuclease H breaks H-bonds of RNA
- DNA polymerase added to replace RNA with DNA
- DNA ligase added to produce double stranded cDNA which is then cloned into vectors
How does high salt concentration disrupt hydrogen bonds?
Salt ions have a higher affinity for the H+/OH- than other water molecules thus binding favours these over hydrogen bonds
What are the 4 enzymes required for creation of cDNA from mRNA?
Reverse transcriptase, Ribonuclease H, DNA polymerase, DNA ligase
How do the clones in a cDNA library vary?
They each contain a different cDNA molecule
What is a contig?
A set of overlapping fragments that represent a DNA consensus sequence
How is DNA isolated from eukaryotic cells (11)?
- Cells mixed with agarose and placed in mould
- Cell wall is ruptured (in agarose)
- Restriction enzyme added (digests DNA)
- Each mould placed in agarose gel well
- Agarose gel run, viewed under UV and ~100,000bp DNA excised
- DNA eluted from excised agarose
- ligated to plasmid vector (BAC) cut w/ same RE
- Treated with DNA ligase
- BAC transformed into bacteria
- bacteria picked into 384 well plate
- DNA isolated for sequencing
What is an alternative name for Sanger sequencing?
Dideoxy chain termination
Describe the process of Sanger sequencing
A radioactive primer, DNA polymerase, dNTPs, ddGTP/ddCTP/ddATP/ddTTP (low conc.) is added to the gene to be sequenced. The gene is replicated by the DNA pol. but if it tries to add a ddNTP then the replication will stop, thus depending on which ddNTP is added the reaction will produce a variety of transcripts terminating on all the A bases (for example). Repeating this with each NTP and then carrying out electrophoresis allows the sequence to be determined
How was the human genome sequenced?
DNA contigs were first sequenced then these contigs were linked by sequencing the ends of large DNA fragments
What is shotgun sequencing?
A method of sequencing used for longer DNA strands, DNA is broken up into small segments which are sequenced using Sanger sequencing, this process is repeated to produce multiple overlapping sequences which computer programs then assemble into a continuous sequence
What is the difference between a dNTP and a ddNTP?
A dideoxynucleoside triphosphate has an H instead of OH at the 3’ position (as well as an H at 2’)
What is the difference between automated DNA sequencing and Sanger sequencing?
Automated DNA sequencing combines all the ddNTPs into one capillary but labels each w/ a different fluorescent dye, a computer may then read the fluorescent peaks on the electrophoresis and generate the sequence from there
What are the names of the two methods of rapid DNA sequencing? Which method is cheaper & faster?
Illumina (Illumina is cheaper & faster), 454
How does the 454 method differ from the illumina method?
The 454 uses beads to attach the adaptors/short DNA sequences to and then picks the beads in wells
Describe the process of the illumina method of DNA sequencing (4)
- Fragment DNA into small 200bp sequences (w/ restriction enzymes)
- Add different adaptors on either end, H-bond these to primers on a glass slide
- Amplify each bound DNA sequence (fragment bends over & H-bonds w/ complimentary primer on slide, DNA polymerase is added to copy the fragment - PCR on a slide)
- DNA is made single stranded, primer added, tagged dNTPs added - as each new base is added by the DNA pol a laser flashes and causes the bases to fluoresce thus determining the base sequence
What are the (2) advantages and (2) disadvantages of the illumina method?
adv: cheap, quick
dis: small fragments, scientists cannot read
How many genes does the human genome code for? How many does the mitochondrial genome?
20,000genes, 37 genes (big difference)
From which parent are the mitochondrial genes inherited?
Mother
Describe the structure of the mitochondrial genome (3)
Circular, heavy(28genes, G rich)/light (9genes, C rich) strands, genes (of H&L) do not overlap
What are the 5 main types of RNA?
mRNA, rRNA, tRNA, snRNA (small nuclear), snoRNA (small nucleolar)
How is the percentage of coding DNA in a gene affected by the gene size?
As gene length increases, the percentage of DNA coding for the protein produced decreases
What is the longest human gene?
Dystrophin, 2,600kBp
What are gene families and how are they formed?
A set of several similar genes formed by the duplication of one original gene
Give 2 human examples of gene families
- alpha/beta globin - code for haemoglobin subunits, differentially expressed through development
- Histone - H1, H2A, H2B, H3, H4, required for DNA replication to keep DNA together/package, each histone family is identical
What is the cause of the symptoms of alpha-thalassemia?
Impaired production of alpha chains from the alpha globin genes leads to impaired oxygen delivery to tissues
How much of our DNA is noncoding? What 5 things make up noncoding DNA?
90% is noncoding! e.g. Introns, regulatory regions, pseudogenes (old genes which do not code - no promoter), gene fragments, microRNAs
What is present in the 5’ flanking sequence of a regulatory gene (2)?
Promoter, enhancer
What 4 things are present in a regulatory gene (not including promoter/enhancer)?
5’ flanking sequence, Intron, Exon, 3’ flanking sequence
What is a processed pseudogene?
A pseudogene where transcription, splicing and polyadenylation occur, but then reverse transcription and re-integration so leads to no introns
Why do the number of genes you have matter?
Important for health to make the correct amount of protein
How did pseudogenes arise?
From gene duplication - pseudogene picks up mutations and loses function
What is the most abundant sequence in human genome?
Alu repeat family, occurs 750,000 times (every 4kb), alu insertions implicated in cancer, primate specific
What is a SINE and a LINE?
SINE: short interspersed nuclear element, LINE: long interspersed nuclear element
Both are pieces of foreign DNA that have been inserted into chromosomes of eukaryotes by foreign agents e.g. retroviruses
Give an example of a SINE
Alu repeat
What 4 things are required for LINE transposition?
promoter, reverse transcriptase (copy RNA to DNA), endonuclease (cleave target DNA), RNase H (RNA removal)
What is a L1 retrotransposon?
A class/type of LINE
Describe the life cycle of the L1 retrotransposon (8)
- Transcription of LINE by host RNA pol.
- Translation of ORF1 & 2
- ORF1 & 2 bind to poly A tail of LINE & move it back into nucleus
- poly A tail of LINE H-bonds with target DNA stretch
- LINE endonuclease cleaves target DNA, reveals a 3’OH for new strand synthesis
- LINE reverse transcriptase copies LINE mRNA into cDNA
- LINE RNase H degrades RNA strand, host DNA pol replaces strand and ligase joins backbone
- LINE integrated into genome
How doe the LINE length vary with age?
Gets shorter with age, LINES truncated on 5’ end, eventually promoter removed (on 5’ end) so LINE is no longer transcribed
Are truncated LINES transcribed?
NO, prick
What may be the consequence of LINE insertion into genome?
- Insertion into promoter can silence a gene
2. Insertion into an intron can slow transcription
Where is LINE transposition more common, germline or somatic cells?
Germline is more common
