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What are restriction enzymes?
Endonucleases with the ability to cleave dsDNA at specific sequences; essential to cloning
How do bacteria protect themselves against restriction enzymes?
By methylated the region which would be targeted by the restriction enzyme; this helps them to only cleave DNA of other cells
What enzymes are used in cloning?
- Restriction enzyme
- DNA ligase: joins DNA fragments
- Exonucleases: clip ss overhangs generated by restriction enzymes for blunt-end joining by ligase
- DNAP I “Klenow” fills in overhangs to generate blunt ends; incorporates radioactive dNTPs into DNA to produce radio-active probes for blots
- Polynucelotide kinase: adds phosphate group to 5’ hydroxy terminus of polynucleotides to generate radioactive probes
What is the most commonly used cloning vector?
Plasmids; this is the smallest sized vector, and has limited carrying capacity (to 15kb), if you want to add more material than 15kb you must choose a larger vector
How do you pick a plasmid/vector?
You choose based on the size of the DNA insert and what experimental questions are being asked
Lambda phage cloning vector
Plasmid/phage hybrid vector; 25-45kb
Bacterial artificial csomes
BAC; 100-300kb; often used for human DNA as our genes are huge
Yease artificial csome
Tricks yeast into replicating the YAC as a real csome; 100-1000kb
Why is the ampicillin resistance gene used in DNA recombination?
It is used to determine which cells have incorporated the plasmid into their genome; only the transformed cells will have ampicillin resistance and only the transformed cells will grow in culture
How do you amplify recombinant plasmids in E. coli?
Put the DNA in liquid media; shake the cells overnight; they will divide over and over
Why is gel electrophoresis needed in gene recombination?
It is needed to confirm that the DNA fragment is correctly inserted into the plasmid vector; you first digest with restriction enzyme followed by gel electrophoresis; the DNA fragment spanning the cloning site containing the inserted DNA generated by the recombinant plasmid will be larger by the size of the DNA inserted than the one produced by the vector
How does cloning using a phage vector work?
- 1. Construct recombinant phage DNA containing inserted DNA to be cloned
- 2. Package recombinant phage vector in phage head proteins to generate infectious phage particles
- 3. Infect bacterial host cells with packaged phage and grow culture 12-15 hr
- 4. Harvest phage particles from lyzed bacteria
- 5. Large quantity of cloned human gene of interest can be purified from the recombinant phage DNA
How do you construct a genomic library using YAC vector?
Use EcoRI for partial digestion of Human DNA to create large csome fragments with sticky ends; take a YAC vector and cut it using EcoRI and Bam HI, to create two fragments with sticky ends; using recombination and DNA ligase you create a YAC with inserted human DNA; this is inserted into a yeast, and the yeast is amplified
What is the difference between a genomic DNA library and a complementary DNA library?
A genomic DNA library is a collection of all the genetic material of an organism, cloned in a vector; a cDNA library is a collection of the expressed genes of a eukaryotic organism cloned in a plasmid vector
How do you produce protein from a cloned gene?
You need a promoter with a -10 and -35 region; then you need signals to ensure translation of the mRNA (Shine-Delgarno sequence, translational start and termination codons)
Can you express genes in a plasmid vector in eukaryotic cells?
No, you must construct a eukaryotic expression vector for efficient expression of the gene in eukaryotic host cells; this vector must contain a gene, a SV40 simian virus intron, poly A signal because the mRNA needs these to transcribe; bacterial origin of replication for amplifying the copy number of the plasmid, ampicillin resistance, SV40 enhancer/promoter for efficient transcription of the selectable marker gene in transfected cells, and neomycin resistance (to allow the small amount of transfected cells to grow in media)
What is transfection?
Purified DNAs of expression are introduced to the chosen eukaryotic cells for functional studies of gene activities or for obtaining protein products in large quantities
What are the methods of transfection?
Electroporation, liposomes, and recombinant viruses
What is electroporation?
You mix the plasmid DNA and apply a high voltage electric field, this pokes holes in the cell membrane so the plasmid can get in; 10-20% of cells are transfected, therefore you need some type of antibiotic resistance to select for transfected cells
How do you use liposomes for transfection?
Enclose the plasmid DNA in a lipid membrane so it can fuse with the host cells membrane; has 10-20% efficiency
How do you use recombinant viruses for transfection?
You package plasmid DNA into viruses to infect the host cells; most commonly used vector is HIV, but you can take out all the viral portions and just use the portions for transcriptional elements; 50-100% of cells infected; most challenging method of transfection; used in gene therapy
How do you use reporter gene assay to locate enhancers?
If you have a gene which you think is a very important suppressor gene (gene X), then you want to determine where its enhancer is, you do this through DNase hypersensitivity mapping (enhancers contain DNA sequence motifs that can bind transcription factors which have a DNA binding domain which can compete with histones for binding, so they are packed loosely in nucleosomes and are accessible for exogenously added DNase, indicated it is potentially an enhancer site)
How do you test a region for enhancer activity?
- 1. Reporter gene assay; In a eukaryotic expression vector, link the 10 kb of DNA spanning the putative enhancer to a reporter gene, whose protein product can be easily detected by enzyme activity
- 2. Make a mutant reporter gene clone with the putative enhancer region deleted
- 3. Separately transfect the normal and the deletion mutant clones into cultured human mammary cells
- 4. Assay reporter gene activity to determine if deletion of the putative enhancer causes a drastic decrease in expression of the reporter gene
How was the human genome originally sequenced?
Chain termination method; the DNA to be sequenced (BAC or YAC clones) is denatured to separate the two complementary strands and mixed with primers (labeled so you know the beginning of the sequence), four dNTPs, dideoxy NTPs with fluorescent tags (these terminate the chains, and each one has a different marker), and DNA polymerase; the sequence of the newly synthesized, fluorescent DNA molecules after gel electrophoresis is read by an automatic fluorescence detector
DNA sequencing by Sanger’s chain termination method
What is massively parallel DNA sequencing?
First you shear the DNA into small lengths, you bind each DNA to a surface and amplify the DNA, drop each bead into a well in a flow cell, then you flow the wells with dNTPs sequentially, with each one having a different marker, this allows you to get the specific sequence; a high resolution camera records the sequentially incorporated nucleotides; each DNA template in a well can be accurately copied for ~200 nt; computer software compares the sequence against a reference genome to determine where it is from
What does PCR require?
DNA template, synthetic primers ~20 bps long, heat-stable DNAP, dNTPs, and an automated thermocycler
Why do Southern blot?
See if a DNA fragment is present in a genome or plasmid
How do you do a Southern blot?
- 1. DNA (genomic or plasmid) is fragmented by restriction enzyme (RE) digestion; the size distribution of the digested DNA fragments depends on the distriburion of the enzyme cleavage sites in the DNA
- 2. The DNA is run through gel electrophoresis, denatured by alkali treatment, then transferred from the gel to a nylon filter the gel
- 3. A radioactively labeled nucleic acid probe, whch is a small fragment of the gene cloned in a plasmid vector) is added and binds to complementary DNA segments
- 4. The nylon is covered by an X-ray film, and after development the positions of the probe become visible
What are the applications of Southern blot?
RFLP, VNTR, and DNase hypersensitivity mapping
What are Northern and Western blot?
Northern blot is to detect RNA, and Western blot is to detect proteins (you use antibodies instead of labeled probes)
What is RFLP?
Restriction fragment length polymorphism; polymorphisms can be single-base substitutions or deletions or insertions altering the number of copies of short stretches of DNA
How do you use RFLP to genotype sickle cell disease patients?
Use it to determine whether a patient has sickle cell or if they are a carrier; DNA cleaved with Mst II, in sickle cell patients MstII cannot cleave the site that it can in normal patients; after Souther blotting and hybridization homozygous patients produce one large band and 1.35kb, while heterozygous patients produce a normal band at 1.35kb and 1.15kb
What is VNTR?
Variable number tandem repeats; short nucleotide sequences ranging from 14 to 100 bases long that are organized into clusters of tandem repeats; these are on many sites on all csomes; these repeat regions can be cleaved out and Southern blot is performed to visualize this; each variant of number of repeats is an allele
What are transgenic animals?
Animals carrying integrated human genes
Why are transgenic animals generated?
To complement studies of human genes, and their enhancers and promoters, in expression plasmids transfected into immortalized and transformed cell lines of various human tissues
How do transgenic animals measure expression of genes?
Expression of genes in transgenic animals measures the true physiological activities of the genes and their regulatory elements
What are knock-out animals and what do they allow scientists to study?
They are animals, mostly mice, which have mutated or deleted genes; they are used to study the effects of the disruption of a gene