Biotechnology today and yesterday

Molecular genetics

Molecular genetics is a subfield of genetics that focuses on the structure and function of genes on a molecular level,

including genetic variation, gene expression, and DNA replication and repair.

This field aims to understand how genes are transmitted from one generation to the next and how they

influence human behavior, health, and disease. Research in molecular genetics relies heavily on laboratory

methods and technologies, such as DNA sequencing, PCR, and gene editing techniques.

Molecular Genetics Methods

1. 1. Polymerase Chain Reaction (PCR)
2. 2. DNA sequencing (manual/automated)
3. 3. DNA Fingerprinting (DNA
4. typing/profiling)
5. 4. Single nucleotide polymorphisms (SNPs)
6. practical applicationsAmplify DNA for Cloning (PCR)

✓ Amplify DNA for sequencing without cloning (PCR)

✓ DNA sequencing reaction (PCR)

✓ Mapping genes and regulatory sequences

• ✓ Linkage analysis (identify genes for traits/diseases)
• ✓ Diagnose disease
• ✓ Pathogen screening
• ✓ Sex determination
• ✓ Forensic analysis
• ✓ Paternity/maternity (relatedness)
• ✓ Behavioral ecology studies (relatedness)
• ✓ Molecular systematics and evolution (comparing homologous
• sequences in different organisms)
• ✓ Population genetics (theoretical and applied)
• ✓ Physiological genetics (studying basis of adaptation)
• ✓ Livestock pedigrees (optimize breeding)
• ✓ Wildlife management (stock identification/assessment)
• ✓ Detection of Genetically Modified Food (GMOs)
• the Polymerase Chain Reaction (PCR)

✓ Ability to generate identical high copy number DNAs made possible

in the 1970s by recombinant DNA technology (i.e., cloning).

✓ Cloning DNA is time consuming and expensive (>>$15/sample).

✓ Probing libraries can be like hunting for a needle in a haystack.

✓ PCR, “discovered” in 1983 by Kary Mullis, enables the amplification

(or duplication) of millions of copies of any DNA sequence with

known flanking sequences.

✓ Requires only simple, inexpensive ingredients and a couple hours.

DNA template

Primers (anneal to flanking sequences)

DNA polymerase

dNTPs

Mg2+

Buffer

✓ Can be performed by hand or in a machine called a thermal cycler.

✓ 1993: Nobel Prize for Chemistry

How PCR works:

1. Begins with DNA containing a sequence to be amplified and a pair

of synthetic oligonucleotide primers that flank the sequence.

2. Next, denature the DNA to single strands at 94˚C.

3. Rapidly cool the DNA (37-65˚C) and anneal primers to

complementary s.s. sequences flanking the target DNA.

4. Extend primers at 70-75˚C using a heat-resistant DNA

polymerase such as Taq polymerase derived from Thermus

aquaticus.

5. Repeat the cycle of denaturing, annealing, and extension 20-45

times to produce 1 million (220)to 35 trillion copies (245) of the

target DNA.

6. Extend the primers at 70-75˚C once more to allow incomplete

extension products in the reaction mixture to extend completely.

7. Cool to 4˚C and store or use amplified PCR product for analysis.

Example thermal cycler protocol used in lab:

Step 17 min at 94˚C Initial Denature

Step 245 cycles of:

20 sec at 94˚C Denature

20 sec at 52˚C Anneal

1 min at 72˚C Extension

Step 37 min at 72˚C Final Extension

Step 4Infinite hold at 4˚C Storage

DNA Sequencing

✓ DNA sequencing = determining the nucleotide sequence of DNA.

✓ Developed by Frederick Sanger in the 1970s.

Manual Dideoxy DNA sequencing-How it works:

1. DNA template is denatured to single strands.

2. DNA primer (with 3’ end near sequence of interest) is annealed to

the template DNA and extended with DNA polymerase.

3. Four reactions are set up, each containing:

1. DNA template

2. Primer annealed to template DNA

3. DNA polymerase

4. dNTPS (dATP, dTTP, dCTP, and dGTP)

4. Next, a different radio-labeled dideoxynucleotide (ddATP, ddTTP,

ddCTP, or ddGTP) is added to each of the four reaction tubes at

1/100th the concentration of normal dNTPs.

5. ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with

normal dNTPS, and produce no phosphodiester bond.

6. Whenever the radio-labeled ddNTPs are incorporated in the chain,

DNA synthesis terminates.

7. Each of the four reaction mixtures produces a population of DNA

molecules with DNA chains terminating at all possible positions.

8. Extension products in each of the four reaction mixutes

also end with a different radio-labeled ddNTP

(depending on the base).

9. Next, each reaction mixture is electrophoresed in a

separate lane (4 lanes) at high voltage on a

polyacrylamide gel.

10.Pattern of bands in each of the four lanes is visualized

on X-ray film.

11.Location of “bands” in each of the four lanes indicate

the size of the fragment terminating with a respective

radio-labeled ddNTP.

12.DNA sequence is deduced from the pattern of bands in

the 4 lanes.

Automated Dye-Terminator DNA Sequencing:

1. Dideoxy DNA sequencing was time consuming, radioactive,

and throughput was low, typically ~300 bp per run.

2. Automated DNA sequencing employs the same general

procedure, but uses ddNTPs labeled with fluorescent dyes.

3. Combine 4 dyes in one reaction tube and electrophores in

one lane on a polyacrylamide gel or capillary containing

polyacrylamide.

4. UV laser detects dyes and reads the sequence.

5. Sequence data is displayed as colored peaks

(chromatograms) that correspond to the position of each

nucleotide in the sequence.

6. Throughput is high, up to 1,200 bp per reaction and 96

reactions every 3 hours with capillary sequencers.

7. Most automated DNA sequencers can load robotically and

operate around the clock for weeks with minimal labor.

DNA Fingerprinting (DNA typing/profiling)

✓ No two individuals produced by sexually reproducing organisms

(except identical twins) have exactly the same genotype.

Why?

✓ Crossing-over of chromosomes in meiosis prophase I.

✓ Random alignment of maternal/paternal chromosomes in

meiosis metaphase I.

✓ Mutation

✓ DNA replication errors (same effect as mutation)