6.3 Advanced Molecular Biological Techniques

1. Polymerase chain reaction (PCR)

2. Restriction fragment length polymorphism (RFLP)

3. DNA sequencing

Polymerase Chain Reaction (PCR)4.4.1: Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA. [Obj. 2]

Until the late 1980s, many copies of a desired DNA fragment could only be made by inserting the DNA sequence into plasmids

Problem: The plasmids had to be extracted from bacteria, and then the desired DNA fragment had to be excised

Solution: Direct method of making copies of a desired DNA sequence, called polymerase chain reaction (PCR) – Kary Mullins, 1985

Polymerase Chain Reaction (PCR)

PCR: Amplification of DNA sequence by repeated cycles of strand separation and replication

Small sample of DNA can be amplified to make multiple copies of a desired DNA fragment

Each PCR cycle doubles the copies of a desired DNA fragment, resulting in exponential growth– ie. after 30 cycles, > 1 000 000 000 copies (230) are made

http://users.ugent.be/~avierstr/principles/pcrcopies.gif

Polymerase Chain Reaction (PCR)One cycle:1. Double-stranded DNA is denatured using

heat (94oC–96oC) to separate strands by breaking hydrogen bonds

• No DNA helicase or DNA gyrase

2. DNA primers (5’-3’) anneal to complementary template DNA that bracket the desired DNA sequence (50oC–65oC)

• No RNA primer

3. Taq polymerase add complementary nucleotides to synthesize the new DNA strand (72oC)

• No DNA polymerase III

Repeat cycle (steps 1-3)

http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

http://croptechnology.unl.edu/animationThumbnails/1020458324.gif

PCR: Length of DNA strands Targeted DNA sequence is not completely

isolated in the first few cycles of PCR

Variable-length strands: Mixture of replicated DNA strands of unequal length– After first cycle, variable-length strands start at

target region on one end and extends beyond the target region on the other end

Constant-length strands: Mixture of replicated DNA strands of equal length– After second cycle, two of the replicated

strands start at target region on one end and terminates at target region on the other end

– By third cycle, number of copies of targeted DNA strands increases exponentially

www.maxanim.com/genetics/PCR/PCR.htm

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro15.swf::Polymerase Chain Reaction

Restriction Fragment Length Polymorphism (RFLP) Polymorphism

– any difference in DNA sequence (coding or non-coding) that can be detected between individuals

Restriction Fragment Length Polymorphism Analysis – technique that compares different lengths of DNA

fragments produced by restriction endonucleases to determine genetic differences between individuals by using complementary radioactive probes

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio20.swf::Restriction Fragment Length Polymorphisms

Restriction Fragment Length Polymorphism Analysis

1. Digest DNA using restriction enzyme(s)

2. Run digested DNA on gel using gel electrophoresis

• Smear - Many DNA fragments with slight differences in length

3. Expose gel to a chemical to denature double-stranded DNA to become single-stranded

4. Southern blotting

RFLP Analysis4. Southern blotting:

i. Transfer DNA from gel to nylon membraneii. Expose nylon membrane to solution with

radioactive complementary nucleotide probes that hybridize to specifically chosen DNA sequences on nylon membrane

iii. Place nylon membrane against X-ray film, where hybridized radioactive probes cause exposure of X-ray film, producing an autoradiogram

http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio_g.swf::Southern Blot

RFLP analysis Differences in

pattern to detect polymorphisms

Animation

DNA Sequencing

Determine sequence of base pairs for genes

Sanger dideoxy method – DNA sequencing technique based on DNA replication using dideoxynucleoside triphosphate

http://www.sanger.ac.uk/Info/Intro/gfx/fred_bw.jpg

Sanger dideoxy methodInto 4 reaction tubes, add:• Double-stranded DNA to be sequenced is denatured to

become single-stranded• Radioactively labelled primer to end of the DNA

template• DNA polymerase• Free nucleotides (dATP, dTTP, dGTP, dCTP)

Into each of the 4 reaction tubes, add a different radioactively labelled dideoxy analogue (nucleoside triphosphate that has no hydroxyl group on the 2’ and 3’ carbon of ribose sugar)

Sanger dideoxy method If dideoxy analogue is missing 3’-OH

on the deoxyribose sugar, DNA polymerase cannot add the next complementary basesynthesis stops

Chain termination resulting in different DNA fragment lengths

Separate different DNA lengths by gel electrophoresis, loading each reaction tube in a separate well/lane

Sequence can be read from the gel in ascending order

http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

Sanger Method Animation

http://www.mefeedia.com/watch/21777157

Human Genome Project To determine the genetic sequence of the

46 human chromosomes Used similar sequencing technique, but

used fluorescently tagged ddNTPs that could be read by a computer

4.4.6: Outline three outcomes of the sequencing of the complete human genome. [Obj. 2] It is now easier to study how genes influence

human development.  It helps identify genetic diseases. It allows the production of new drugs based

on DNA base sequences of genes or the structure of proteins coded for by these genes.

It will give us more information on the origins, evolution and migration of humans. 

4.4.11: Define clone. [Obj. 1]

Clone: a group of genetically identical organisms or a group of genetically identical cells derived from a single parent cell. 

http://www.dnalc.org/resources/animations/cloning101.html

http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/

Steps for cloning a gene: http://highered.mcgraw-hill.com/olcweb/cgi/

pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Steps in Cloning a Gene

4.4.12: Outline a technique for cloning using differentiated animal cells. [Obj. 2]

http://www.massasoit-bio.net/courses/136/136_courseassets/cummings_animations/cloning.html

4.4.13: Discuss the ethical issues of therapeutic cloning in humans. [Obj. 3]

Source: http://www.ibguides.com/biology/notes/genetic-engineering-and-biotechnology