Week 3: Recombinant DNA

DNA containing genetic sequences from multiple sources, assembled in a way not found in nature

Vector

Specially engineered genetic construct

Often in form of small circular pieces of DNA called plasmids

Cloning

Restriction Enzymes: DNA cutting enzymes (molecular scissors)

Plasmid DNA Vectors Enzymes: DNA-cutting circular form of self replicating DNA

Restriction Enzymes

Primarily found in bacteria

Cut DNA by cleaving phosphodiester bonds

Bind to, recognize, cut DNA within specific sequences called restriction site —- palindromes

RE Sticky/Cohesive vs Blunt Ends

Sticky/C: cut DNA with overhanging single stranded ends — advantageous as easily joined together with base pair of complementary bases on other sticky ends

B: double stranded ends

Plasmid DNA

Small, circular pieces of DNA primarily found in bacteria

Extrachromosomal DNA, in cytoplasm

Replicate independently of chromosome

Can be vectors = VERY IMPORTANT FOR CLONING

Recombinant DNA Advisory Committee

1975 — RAC

Evaluate technology and establish guidelines

Transformation

Process of causing bacterial cells to uptake and express exogenous DNA (transfection when used in context of eukaryotic cell types)

Cell must be made “competent” to acquire DNA through diffusion through plasma membrane

Some bacteria re naturally competent

Selection

Process designed to facilitate identification of recombinant bacteria while excluding bacteria that do not contain desired DNA

Primarily through Antibiotic selection

Cloning Simplified

1. cut and join DNA fragments

2. Insert DNA into a plasmid (insert DNA)

3. Produce large amounts of insert DA by allowing bacteria to replicate the recombinant DNA

First ones were insulin and growth hormone

A good vector

Size: small enough to be isolated from chromosomal DNA of host plasmid with chemical techniques (also easier to replicate)

Origin of replication (ori): site for DNA replication

Multiple cloning site (MCS): for several restriction sites, becoming less necessary

Selectable marker genes: often antibiotic resistant genes with bacterial work

RNA polymerase promoter sequences

Cloning a GOI

Genomic gene libraries — random shearing

Complementary DNA libraries — reverse transcriptase

Being phased out

Cloning a GOI using PCR

PCR: Polymerase chain reaction: making copies/amplifying a specific sequence of DNA in a short period of time

1. Target DNA added to tube, mixed with nucleotides (dATP, dCTP, dGTP, dTTP), buffer, and DNA polymerase

2. Paired set of forward and reverse primers are added (complimentary to nucleotides flanking opposite ends of target DNA

3. Reaction tube placed in thermocycler

PCR

Creates many copies of a specific DNA sequence

• Specificity

• Amplification

PCR Components

Template: DNA containing target segment

Primers: typically identical in sequence to the 5’-3’ strand

Nucleotide Triphosphates (dNTPs) - building bloks of newly synthesized DNA polynucleotides

Buffer and Salt components: for pH, mg2+ required, DMSO/betaine alter melting temperature

DNA Polymerase

Extends new strand DNA off of 3’ of primer

Adds nucleotides complementary to template strand

Thermostable in modern PCR

Agarose Gel Electrophoresis

Agarose (From seaweed): semisolid gel with small pores through which DNA will travel

Percentage of agarsoe to buffer solution determines ability to separate DNA fragments by size

Gel Electrophoresis

Separating biomolecules based on size

DNA has overall negative charge due to phosphates on backbone

Smaller pieces migrate to positively charged electrodes faster than larger

Tris-Acetate-EDTA (TAE) required as electrolytes to conduct charge

Imaging a DNA Gel

DNA binding fluourescent dye — intensity depends on amount of DNA bases present

Bands can be compared to markers/ladders of known size

Sanger Sequencing

Gold Standard for Modern Sequencing

DNA polymerase and a primer to replicate a strand of DNA

Also contains ddNTP (dideoxyribonucleotide triphosphate) in addition to normal dNTPs == termination point

Run on “capillary electrophoresis” machines instead of agarose

Confirms sequence of specific span of DNA

Next Generation Sequencing (NGS)

Generates a larger amount of data

Reference genomes, entire genome for comparison, metagenome, sequencing all RNA transcripts collected from an organism (RNAseq), indexing patients/samples

Illumina, PacBio, Nanopore Technologies

Other Techniques

FISH: Fluorescence in situ hybridization — pinpoint gene of interest

Chromosome location and copy number

Southern Blotting: uses a membrane

Microarrays: “gene chips” probes of known sequences attached to a solid surface

Genomics

Cloning, sequencing, and analyzing entire genomes

Bioinformatics

Merging molecular biology with computer technology

Human Genome Project

International effort to identify all human genes and to sequence all the base pairs of the 24 human chromosomes (1990)

Designed to analyze genetic variations among humans, map and sequence genomes of model organisms, develop new lab technology, disseminate genome information, consider ethical/legal/social issues

Published 2003

1. 3.1 million base pairs

2. genome 99% same between nationalities

3. Diversity from single-nucleotide polymorphisms, copy number variations

4. Less than 2% of genome codes for genes

5. Vast majority DNA is non-protein coding, 50% is repetitive sequences

6. 20,000 protein-coding genes

7. Functions of at least 40% human genes still unknown

8. Chromosome 1 = most genes, Y chromosome = fewest

Systems biology

Incorporating data from genomics, transcriptomics, proteonomics, other areas of biology

Interactome, network map