Methods in Molecular Biology

Basics for Molecular Biology

Cloning typically involves the use of E. Coli

Bacteria: single cell; round, cylindrical, or spiral

E. Coli: studied for 100 yrs; grows rapidly (doubling time = 30 min); non pathogenic

Clonal Growth

cell and contents double in size then divides across the middle via binary fission

grown on agar

single cells Þ colony overnight

Plasmids

E. Coli DNA = circular and double-stranded
Plasmids are accessory (extra) circles of DNA
Can take plasmid out, incorporate our DNA and put back in
Vector portion: the part derived from the native bacterial plasmid

Bacteriophages

viruses of bacteria (ie phage l)

attach to E. Coli and infect it

the DNA is replicated, transcribed, translated

new phage are assembled and burst out of the cell

when a cell bursts open, it forms a plaque (clone of phages) Þ easy to replace phage l DNA with our own

can assemble intact phages to infect E. Coli

M13 particle Þ single stranded DNA

Cosmids: plasmids carrying the phage l packaging signal

Phagemids: plasmids carrying the packaging signal of M13

cDNA and Genomic Cloning and Tools for Analysis of Gene Structure and Function

gene structure and flow of genetic info

5' flanking region: contain cis acting elements:

TATA: correct initiation of transcription
CAAT: degree of transcriptional activity
HRE: responsiveness to hormones
TSE: tissue specific expression

MRNA

exons are in mature mRNA and introns are removed via splicing before maturation
gains a cap and a poly A tail

the basic paradigm underlying DNA cloning

3 components: the DNA to be cloned, a DNA vector (sort, amplify, shuttle), and a host
bacteria never takes up more than one plasmid

restriction enzymes facilitate construction of recombinant DNA and subsequent cloning

restrict enzymes = bacterial endonucleases

cleaves any foreign DNA taken up by infection or transformation

usually 4-6 bp long and palindromic with sticky or blunt ends

Genomic Libraries – Construction and Content

Genomic library = total # of bacterial colonies or plaques that collectively contain fragments of DNA for the entire genome

Can Ý the size of cloned fragments by partial digestion which ß colony # and simplifies screening

Lambda phage can handle larger fragments better than plasmids

If looking for a protein coding gene, can simplify process by using a cDNA library

cDNA libraries: Content and Construction

derived from mature mRNA

only protein coding genes are included

mRNA Þ Þ Þ (reverse transcriptase)Þ Þ Þ cDNA

Screening of cDNA and Genomic Libraries by Hybridization

Use a specific probe that will hybridize a cDNA clone

How do you select a specific DNA probe? Þ purify the protein encoded by the gene Þ determine partial a.a. sequence Þ synthesize all potential sequences Þ use as probes

2 important factors: length of probe (oligonucleotide ~12-14 nt) and temp. of hybridization (critical for probe specificity) Class Information:

Cloning

DNA Þ amplify Þ many DNA

Need vector to amplify things in

use plasmid vector (small circular DNA in bacteria): replicate rapidly, multiple copies (~30 to 40), have antibiotic resistence genes, ori = origin of replication, also want selectable marker
screening: can put reporter genes around the cloning site in order to indicate whether the gene has been inserted

if blue = no gene put in
if white = gene incorporated and disrupted the reporter gene

How do you insert the gene?

Restriction enzyme

cut DNA at specific sequence
similar to bacterial immune system

can chop up foreign DNA
defense mechanism
will methylate sequence to determine what is self and what is foreign

EcoRI gives very defined cut; if methylated, won’t cut
many restriction sites in the MCS for many enzymes, thus can buy the enzyme, cut the MCS, and insert the gene

Transformation

Other ways to clone

Sticky ends Þ very high efficiency (see above)

Blunt ends (Dra I restriction enzyme)

bacteria is the host cell: put DNA into a host cell (vector)

can have 4 base cutter, 6 base, 8 base, etc

ACGT – 4 Þ 4⁴ = every 256 bp

GAATTC – 6 Þ 4⁶ = every 4096 bp

4⁸ = every 65,536; etc

cDNA and genomic DNA differences

Vector Types

Plasmids: small, circular DNA’s Þ form colonies

insert size 5 to 10 kb; transformation (low efficiency)

l phage (virus that infects bacteria) Þ bacteriophage

linear DNA Þ form plaques; infection (high efficiency)

size ~48 kb; insertable size ~20kb

cosmids

plasmids with l signals in them

20 to 40 kb; infection (high efficiency)

YACs (yeast artificial chromosome)

add yeast origin, centromere, 2 telomere

insert size £ 10⁶ kb

Library Construction

Genomic DNA library: take total genomic DNA Þ cut into pieces via restriction enzymes or random shearing or partial digestion (form overlapping fragments)

cDNA library (want for brain): take brain tissue and digest it Þ rRNA (90%), tRNA (5 to 10%), mRNA (1 to 5%) Þ centrifuge to isolate mRNA Þ use poly A tail to help select out mRNA

next, use reverse transcriptase to form cDNA Þ add on sticky ends and put into vectors

Screening

Make protein from the cDNAs and screen the expression library with Ab Þ lots of problems involved

Screen using DNA: rare – globin; using homologous proteins (can use to pull out a small piece of our gene)

Use degenerate probes

Now we have a probe

We now have a plate full of plaques Þ replicate it then store original plate Þ nitrocellulose or nylon Þ denature the DNA on the copy Þ anneal probe via hybridization Þ wash to get rid of false positives (changing stringencies – salt and temp)

Chromosome walking

Electrophoresis

Method of separating molecules on basis of charge and size; Can detect using radioactivity

Add glycerol or urea and a dye to your sample; Add SDS Þ coats all similar charge thus separates according to size only

Can detect using radioactivity

Agarose Þ for large DNA/RNA fragments; Acrylamide for small molec wght fragments

Western blot Þ protein Þ detect with Ab; Southern blot Þ DNA Þ transfer to nitrocellulose Þ probe with oligonucleotide;

Northern blot Þ RNA; Can use native gels (DNA) or denatured gels (RNA)

PCR