Lecture 3: Methods in Cell Physiology

recombinant DNA

any DNA molecule composed of sequences derived from different sources

DNA cloning

produces large amounts of purified DNA to study in cells

process of recombinant DNA technology

vector + DNA fragment → recombinant DNA → replication of recombinant DNA within host cells → isolation, sequencing, manipulation of purified DNA fragment

what does PCR stand for

polymerase chain reaction

PCR

amplifies a specific DNA region exponentially

requirements for PCR amplification

DNA template, primers, DNA polymerase, Deoxynucleotide Triphosphates (dNTPs)

primers

short DNA sequences flanking target region

how might amplifying a gene from genomic DNA be problematic

uneven amplification

can PCR be performed on RNA

no

how can complementary DNA (cDNA) be generated

from mRNA using retrovirus enzyme reverse transcriptase

what is compatible with PCR

DNA, single or double stranded cDNA

vector

DNA molecule used as a vehicle to carry foreign genetic material into a host cell

how do you insert the DNA fragment into the vector

with restriction enzymes to cut both the vector and insert, creates sticky ends, then use DNA ligase to put them together

plasmid cloning vectors

enables amplification in host organism

what does ORI stand for

origin of replication

3 elements to plasmid cloning vectors

polylinker region, selectable genre, replication origin sequence

restriction enzymes

bacterial proteins that are used to cut DNA at restriction sites

restriction sites

palindromic sequences (reads the same back and forth)

stick ends

DNA fragment have them to ligate into a vector cut with the same restrictrion enzyme

ligase

joins DNA fragments together if the ends are compatible

vector and DNA fragment complementary base pair via ___

sticky ends

how does DNA ligase join DNA fragments

covalently joins sugar phosphate backbone

PCR primer sequences

used to add restriction sites flanking gene of interest

resulting PCR product is flanked by restriction sites compatible with the vector, what are the 2 restriction sites used in the example

incorrect orientation, vector self-ligation

what happens to the DNA fragment and vector when using restriction enzymes and are ligated together

digested

steps in DNA cloning

vector and DNA fragment are cut with compatible enzymes, cut vector and DNA fragments are mixed with DNA ligase to form recombinant plasmid, E. Coli bacteria are mixed with recombinant plasmid and bacteria internalize the plasmid called transformation, growth media containing antibiotics enables selection of only bacteria that have plasmid, plasmid DNA continues to amplify in selected bacterial cells

bacterial expression vector plasmids commonly use this system

lac promoter system

lac promoter

repressed by Lacl, only expresses lacZ when lactose is present

how does plasmids modify lac promoter system

replacing lacZ with gene of interaction, then using IPTG as mimic of lactose to express gene

transfection

introduction of recombinant DNA into eukaryotic cells using non-viral methods

goal of transfection

express gene as protein in cells

transient transfection

vector expresses gene of interest within the nucleus but is eventually lose as cells divide

stable transfection

vector integrates into the genome and expresses gene of interest as long as the culture is maintained, requiring selection using antibiotics

how does retrovirus infect

by reverse transcribing RNA genome into DNA then integrating into host genome

retroviral vectors

use three-plasmid system to make virus particles that deliver genes of interest to cells

type of plasmids in retroviral vectors

vector plasmid, packaging plasmid, viral coat plasmid

vector plasmid

contains gene of interest, promoter, resistance gene

what does packaging plasmid contain

viral capsid that protects nucleic acid

viral coat plasmid

contains attachment protein to enable the virus to bind and enter cells

key components of CRISPR/Cas9 Gene Editing

guide RNA (gRNA), target sequence, Cas9 protein

how does CRISPR/Cas9 Gene Editing work

gRNA guides Cas9 to target sequence in genome, Cas9 creates a double-stranded break in DNA, cell attempts to repair break

what does repair often lead to

insertions/deletions, gene knockout

DNA cloning step 1

vector and DNA fragment are cut with compatible enzymes

DNA cloning step 2

the cut vector and DNA fragments are mixed with DNA ligase to form recombinant plasmid

DNA cloning step 3

E. Coli bacteria are mixed with recombinant plasmid and bacteria internalize the plasmid in transformation

DNA cloning step 4

growth media containing antibiotics enables selection of only bacteria that have the plasmid

DNA cloning step 5

plasmid DNA continues to amplify in selected bacterial cells

why do vectors carry foreign genetic material into a host cell

for genetic engineering, cloning, gene therapy