本期內容是 DNA 與 分子生物技術，實驗手冊與實驗模擬請看后兩期。本部分內容來自 University of California, Berkeley - UC Berkeley Extension, 虛擬實驗的內容來自 Labster. 本部分內容均不會標記為為原創(chuàng)，但由于是UP主購買的課程，因此不接受非授權的轉載，謝謝您的理解。

每一個生物基礎實驗均會分為三部分：第一部分為實驗的生物理論；第二部分為實驗的指導手冊；第三部分為 Labster 的虛擬實驗模擬。第一部分的基本信息由 Ying Liu, Ph.D. 提供，第二部分的實驗手冊來自 Labster, 第三部分的實驗模擬過程均由UP主操作。

Lab 9 -?Polymerase Chain Reaction

Structure of DNA

- Backbone: S = sugar, deoxyribose; P = phosphate

- Nucleotide bases: A = adenine; C = cytosine; G = guanine; T = thymine

- Base pairing: G-C 3 hydrogen bonds; A-T 2 hydrogen bonds

Chargaff’s rules:

Purines (A+G) = Pyrimidines (T+C)

%G = %C

%A = %T?

Rosalind Franklin (1920 - 1958): produced X-ray crystallography of DNA

James Watson and Francis Crick: discovered the structure of DNA double helix in 1953??

Watson, Crick and Wilkins received Nobel Prize in 1962

Franklin’s contribution has not been properly acknowledged

DNA Replication

When a cell is ready to divide, it goes into S phase to duplicate DNA；

After duplication, the sister chromatids are held together by the centromere, and they carry the same genetic information.

The structure of DNA double helix

DNA Directionality

DNA backbone is formed by phosphodiester bond between the 5’ phosphate- and 3’ hydroxyl-groups of adjacent nucleotides;

DNA double helix is stabilized by hydrogen bonds between the bases, and is antiparallel.

G≡C

A=T

DNA Replication

Base-pairing enables DNA replication；

Each strand can serve as a template for replication；

Semiconservative: each daughter DNA contains one original (old) strand and one newly synthesized (new) strand；

DNA replication takes the careful orchestration of the actions of 30 different enzymes.

DNA Replication: Initiation

Replication origin: AT-rich;

Topoisomerase: relax supercoiled DNA;

Helicase enzyme unwinds part of DNA helix;

Stabilized by single-stranded binding proteins.

DNA Replication: Elongation

Primase synthesizes a RNA primer

DNA polymerase III adds nucleotides to the 3’ end;

Leading strand DNA synthesis is continuous;

Lagging strand uses a ‘back-stitching’ mechanism to synthesize Okazaki fragments, which are later joined together;

DNA ligase links the fragments together.

Energy:

Making Lots of Copies of DNA

Molecular cloning: clone DNA of interest into a bacterial plasmid vector and transform the plasmid into E. coli;

When E. coli multiply, plasmids also multiply.

PCR

PCR (Polymerase Chain Reaction) uses a DNA polymerase to amplify selected DNA sequences in a test tube;

Extremely sensitive: only need 1 cell of DNA to start.

PCR Process

PCR Primers

PCR primers are critical!

Need to know a bit of sequence to make proper primers;

Primers bracket target sequence for DNA amplification.

PCR?Primers

PCR Program

Heat (94oC) to denature DNA strands;

Cool (59oC) to anneal primers to DNA template;

Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA;

Repeat 40 cycles.

PCR reaction usually 30-40 cycles.

Amplify specific region of DNA from a small amount of starting DNA.

PCR: DNA Polymerase

Heat DNA to denature (unwind) it destroys DNA polymerase (enzyme);

Taq polymerase: from hot springs bacteria ;

Kary Mullis developed PCR in 1985, received Nobel Prize in Chemistry in 1993 because it revolutionized the way that DNA and RNA are Analyzed.

PCR Applications

PCR for Forensic Application

RT-PCR: Use reverse transcriptase to convert RNA to DNA to serve as PCR template;

qPCR: quantitative (real time) PCR using fluorescently-labeled primers.

What is in Our Genome?

The human genome is about 3,000 Mb (million base pairs);

(roundworm) has 100 Mb, and Drosophila (fruit fly) has 165 Mb;

has 20,000 genes, and Drosophila has 14,000 genes;

Before the human genome was completely sequenced, researchers predicted that the human genome would contain about 50,000 - 100,000 genes;

The actual number of genes in the human genome is about 21,000.

98.5% of the genome does not code for proteins, rRNAs or tRNAs (non-coding sequence).

About a quarter of the genome codes for introns and gene-related regulatory sequences;

The genome contains large amount of repetitive DNA including transposable elements (transposons, other repetitive sequence, > 50%).

We Are Mostly the Same

It is estimated that individual human genomes are 99.5% the same;

Humans and chimpanzees are 98.8% similar;

Where does our phenotypic variations come from?

- Different combinations of alleles;

- Mutations.

Some parts of the genome are more variable than others (mutation hotspots);

These regions can be Analyzed for individual differences (genetic fingerprint).

Sources of Genetic Variations

SNP: single nucleotide polymorphism;

STR (microsatellite): short tandem repeats (2-6 bp);

VNTR (minisatellite): variable number tandem repeats (10-100 bp).

Short Tandem Repeats

Short tandem repeats (STRs): 2-6 bp;

Copy numbers range from 5-50 or more.

Variable Number Tandem Repeats

Variable number tandem repeats (VNTRs): 10-100 bp;

Copy numbers range from 5-50 or more;

Both STR and VNTR are used for DNA fingerprinting.

Combined DNA Index System (CODIS)

CODIS: US national database, maintained by FBI;

Contains samples from convicted offenders, missing persons, and crime scenes;

CODIS core: 13 loci - In 2017, expanded to 20 loci.

Agarose Gel Electrophoresis

Gel electrophoresis: separates DNA, RNA, proteins and their fragments based on size and charge;

DNA gel: agarose forms matrix, apply electric field, DNA (- charged) moves towards + end;

Smaller fragments migrate faster;

Compare to known size marker to estimate the size of DNA fragments.

Dyes and Staining

Loading Dye (LD): bromphenol blue and xylene cyanol, co-migrate with DNA (~500 bp and ~6 kb, respectively);

Helps you load the samples easily and monitor the progress of electrophoresis;

After the electrophoresis: Fast Blast DNA stain binds to DNA on the gel so you can detect bands.

DNA Size Marker

DNA Size Marker: bacteriophage λ DNA digested with HindIII, producing known sized fragments;

Tells you how big your DNA fragments are (molecular weight of each fragment).

Visualizing the DNA

Stain gels: Carefully slide gel into the staining tray, stain the gels for 2-3 minutes, but not for > 3 minutes;

Rinse gels: Transfer the gel into a large container of clean, warm tap water, gently shake the gel in the water for ~10 seconds;

Destain gels (twice): Transfer the gel into a large container of clean, warm tap water, move the gels gently in the water for 5 minutes;

Record results: Pour off the water and examine the stained gels for DNA bands (take photo).

DNA Fingerprints

DNA fingerprint (genetic profile) is a specific pattern of DNA fragments that is unique to an individual.

Some techniques:

- RFLP Analysis: variation within restriction sites between DNA samples'

- Analysis of many (usually 13) parts of the genome known to be highly variable in the human population (STRs / VNTRs);

- PCR can amplify variable DNA markers from tiny amounts (as few as 20 cells!) of biological sample (hair, saliva, semen, blood, etc.).

Restriction Enzymes (RE, Endonucleases)

Naturally occurring enzymes in bacteria (and archaea): chop up foreign DNA, e.g. from bacteriophage

First isolated in 1970; revolutionized Molecular Biology, and Nobel prize awarded in 1978

3000 now known, 600 ‘off the shelf’

Named after the bacterial strain from which isolated:

- HindIII (from Haemophilus influenzae)

- EcoRI (from E. coli)

Make internal cuts in DNA

Each enzyme recognizes and cuts at particular place in a particular sequence of nucleotides = recognition site (restriction site)

Restriction sites are 4-8 bases long, and are often symmetrical (palindromic)

Restriction enzyme digests generate reproducible numbers of fragments in particular DNA sample

Some restriction enzyme digest creates blunt end, others create sticky end.

RFLP

RFLP: Restriction Fragment Length Polymorphism

RFLPs can detect differences in homologous nucleotide sequences of DNA molecules that occur within restriction sites.

本期內容到此結束，感謝閱讀！下一期為實驗手冊 & 下下期將進行 Labster 實驗！