1. Tools for Molecular Biology
Kim Foreman, PhD
Cancer Center, Room 235
August 13, 2019

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

3. Restriction enzymes cleave DNA at specific sequences5’ 3’
CUT G
FLIP
180º T C A
Palindrome

4. Restriction enzymes cleave DNA at specific sequences
Blunt cut
Sticky ends

5. Restriction enzymes produce DNA fragments that can be easily rejoined

6. Creating recombinant DNA

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

8. Amplification of a specific DNA sequence

9. Recombinant DNA and medicine

10. Gel electrophoresis separates DNA molecules based on size

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

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

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

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

15. 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)

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

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

18. Polymerase Chain Reaction (PCR)

19. PCR Movie

20. 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
I I II II II Controls

21. Microsatellite analysis by PCR

22. Synthesizing DNA copies (cDNA) of RNA

23. Reverse Transcription-PCR for diagnosis of HIV infection

24. 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.

25. Oncotype Dx for breast cancer

26. Oncotype Dx for breast cancer

27. Sequencing DNA

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

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

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

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

32. Traditional sequencing versus NGS
Traditional NGS

33. The Human Genome Traditional Sequencing
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