Gene Cloning, PCR, Sequencing, and CRISPR Cas

Gene cloning

process of producing many identical copies of a specific gene

Recombinant DNA technology

combining DNA from different sources into a single molecule

Cloning purpose 1

produce large quantities of DNA

Cloning purpose 2

express genes and study proteins

Cloning purpose 3

produce medically useful proteins such as insulin

Recombinant DNA

DNA molecule formed from multiple sources

Donor DNA

fragment of DNA containing gene of interest

Vector

DNA molecule used to carry donor DNA into host cell

Plasmid

small circular DNA molecule used as vector in bacteria

Plasmid replication

replicates with bacterial DNA and passed to daughter cells

Vector requirement

must replicate and be maintained in host cells

Restriction enzymes

enzymes that cut DNA at specific sequences

Restriction endonucleases

another name for restriction enzymes

Restriction sites

specific DNA sequences where enzymes cut

Palindromic sequences

sequences that read the same in opposite directions

Sticky ends

overhanging single-stranded DNA ends after cutting

Blunt ends

straight cuts with no overhang

Restriction enzyme origin

produced by bacteria to defend against viruses

Recombinant vector production step 1

restriction enzyme digestion of donor and vector DNA

Recombinant vector production step 2

base pairing of complementary sticky ends

Recombinant vector production step 3

ligation by DNA ligase

DNA ligase

enzyme that seals sugar-phosphate backbone

Recombinant plasmid

plasmid containing inserted donor DNA

Transformation

process of introducing recombinant DNA into bacteria

Competent cells

bacteria treated to take up DNA

Transformation outcome

bacteria replicate recombinant DNA as they divide

Selection

identifies cells that have taken up plasmid

Screening

identifies cells with correct DNA insert

Selectable marker

gene allowing survival under selective conditions

Ampicillin resistance gene

example of selectable marker (beta-lactamase)

Selection mechanism

only cells with plasmid grow on antibiotic plates

Screening method

lacZ gene used for blue-white screening

lacZ gene

encodes beta-galactosidase enzyme

X-gal

substrate that turns blue when cleaved by beta-galactosidase

Blue colonies

contain non-recombinant plasmid (functional lacZ)

White colonies

contain recombinant plasmid (disrupted lacZ)

DNA library

collection of recombinant vectors with DNA fragments

Genomic library

contains fragments of entire genome

cDNA library

contains DNA made from mRNA using reverse transcriptase

Reverse transcriptase

enzyme that synthesises DNA from RNA

PCR (polymerase chain reaction)

method to amplify DNA sequences

PCR function

produces millions to billions of copies of DNA

PCR basis

mimics natural DNA replication

PCR requirement

specific primers flanking target region

PCR components

template DNA, DNA polymerase, dNTPs, primers, buffer, Mg²⁺

Primer

short DNA sequence that initiates DNA synthesis

Primer binding

occurs on opposite strands flanking target DNA

Primer direction

binds 5′ to 3′ providing 3′ OH group

PCR necessity of primers

DNA polymerase cannot initiate synthesis alone

PCR cycle step 1

denaturation (~95°C) separates DNA strands

PCR cycle step 2

annealing (~55–68°C) primers bind to DNA

PCR cycle step 3

extension (~72°C) DNA polymerase synthesises DNA

PCR amplification

each cycle doubles DNA amount

Thermostable DNA polymerase

enzyme stable at high temperatures

Taq polymerase

DNA polymerase from Thermus aquaticus

Taq property

remains active at high temperatures

PCR breakthrough

use of thermostable polymerase enabled automation

PCR inventor

Kary Mullis (1983)

PCR advantage

fast, specific, and inexpensive

Gel electrophoresis

technique to separate DNA fragments

DNA movement in gel

moves toward positive electrode

Separation basis

fragment size

Fragment speed

smaller fragments move faster

DNA band

represents fragments of specific length

Gel extraction

fragments can be removed for analysis

Sanger sequencing

method to determine DNA sequence

Dideoxy sequencing

another name for Sanger sequencing

ddNTPs

chain-terminating nucleotides lacking 3′ OH

ddNTP function

stop DNA synthesis when incorporated

Sequencing principle

produces fragments of different lengths

Sequence determination

fragments separated by gel to read sequence

Next-generation sequencing

modern high-throughput sequencing methods

NGS advantage

faster and can sequence entire genomes

CRISPR

clustered regularly interspaced short palindromic repeats

Cas proteins

CRISPR-associated DNA-cutting proteins

TracrRNA

trans-activating CRISPR RNA

CRISPR function in bacteria

adaptive immune system against viruses

CRISPR mechanism bacteria

stores viral DNA fragments in genome

CRISPR memory

stored sequences allow recognition of future infections

CRISPR RNA

guides Cas protein to matching viral DNA

Cas enzyme function

cuts and destroys viral DNA

CRISPR-Cas9

gene editing system adapted by scientists

Cas9

DNA-cutting endonuclease

Guide RNA (gRNA)

directs Cas9 to specific DNA sequence

CRISPR action

creates double-strand break in DNA

DNA repair after CRISPR

cell repairs break using NHEJ or HDR

NHEJ (non-homologous end joining)

error-prone repair introducing mutations

HDR (homology-directed repair)

precise repair using template DNA

Gene editing outcomes

gene knockout, modification, or replacement

CRISPR advantages

highly specific, efficient, faster, cheaper than older methods

CRISPR applications

research, biotechnology, medicine, agriculture