Tools for Molecular Biology Kim Foreman, PhD kforema@luc.edu Cancer Center, Room 235 August 13, 2019

Lecture outline Restriction enzymes Recombinant DNA Gel electrophoresis Nucleic acid hybridization / Southern blot Polymerase chain reaction (PCR) Microsatellite analysis Microarray analysis Sequencing

Restriction enzymes cleave DNA at specific sequences 5’ 3’ CUT G FLIP 180º T C A Palindrome

Restriction enzymes cleave DNA at specific sequences Blunt cut Sticky ends

Restriction enzymes produce DNA fragments that can be easily rejoined

Creating recombinant DNA

Insertion of a DNA fragment into a bacterial plasmid with DNA ligase

Amplification of a specific DNA sequence

Recombinant DNA and medicine

Gel electrophoresis separates DNA molecules based on size

Standard agarose gel Polyacrylamide gel (small DNA pieces) Pulse Field (large DNA pieces)

Complementary nucleic acid strands can bind each other by a process called hybridization

Southern blotting can detect a specific DNA molecule within a complex mixture

Southern blotting can detect a specific DNA molecule within a complex mixture

Detection of Sickle-cell disease by RFLP analysis Type of hemoglobin A – Pro – Glu – Glu – – CCT–GAG–GAG – MstII site – Pro – Val – Glu – – CCT–GTG–GAG – S MstII MstII MstII β-hemoglobin gene 1.1 kb 0.2 kb 1.3 kb Probe for Southern blot Mother Father Child Fetus (AS) (AS) (SS) (AA) Genotype Southern blot of genomic DNA cut with MstII βS (1.3 kb) βA (1.1 kb)

In situ hybridization can locate specific genes on chromosomes FISH: Fluorescent in situ hybridization

Amplification of the Her2 gene in breast cancer Her2 Cep17 Her2 Cep17 No Her2 amplification Her2 amplification

Polymerase Chain Reaction (PCR)

PCR Movie

Diagnosis of Hemophilia by PCR Design Factor VIII gene 10 kbp Site of Bcl I mutation in intron 18 Fragment amplified by PCR 142-bp Pedigree Analysis Fragments 142 bp Bcl I site Absent Bcl I site present Results 99 bp 43 bp 1 2 3 4 5 6 7 I-1 I-2 II-1 II-2 II-3 Controls

Microsatellite analysis by PCR

Synthesizing DNA copies (cDNA) of RNA

Reverse Transcription-PCR for diagnosis of HIV infection

Microarrays can measure the expression of many genes at the same time Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA microarray – a slide on which copies of single-stranded DNA fragments representing an organisms genes are fixed. Each spot represents a different gene.

Oncotype Dx for breast cancer

Oncotype Dx for breast cancer

Sequencing DNA

DNA sequencing detects a 3-bp deletion in the CFTR gene in cystic fibrosis.

Next Generation Sequencing Illumina HiSeq platform – “sequence by synthesis”

Next Generation Sequencing Illumina HiSeq platform – “sequence by synthesis”

Next Generation Sequencing Illumina HiSeq platform – “sequence by synthesis”

Traditional sequencing versus NGS Traditional NGS

The Human Genome Traditional Sequencing 2000-2003 first human genome sequenced 13 years $2.7 billion Sequencing Watson’s genome (NGS) 2007 4 months $1.5 million Illumina (NGS) 8 days $10,000 MinION (Oxford Nanopore Technologies) less than $1000 genome 15 minutes